Brain Research, 597 (1992) 155-161
155
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25436
Single retinal ganglion cells sending axon collaterals to the bilateral superior colliculi: a fluorescent retrograde double-labeling study in the Japanese monkey ( Macaca fuscata) Yasuko Kondo
a, M a s a h i k o T a k a d a b, T e t s u r o K a y a h a r a c, Y u k i h i k o Yasui c, K a t s u m a N a k a n o c and Noboru Mizuno b
Departments of b Morphological Brain Science and a Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto (Japan) and c Department of Anatomy (1st Division), Mie Unicersity School of Medicine, Tsu, Mie (Japan) (Accepted 18 August 1992)
Key words: Visual system; Retinal ganglion ceil; Retina; Superior colliculus; Retinotectal projection; Bilateral projection; Axon collateralization; Monkey
Single retinal ganglion cells projecting bilaterally to the superior colliculi (SC) by way of axon collaterals were revealed in the Japanese monkey
(Macaca fuscata). After injecting Fast blue into the SC on one side and Diamidino yellow into the SC on the opposite side, some retinal ganglion cells were double-labeled with both tracers. Most of them were large cells (more than 25 ~m in diameter), and were localized in a narrow strip around the vertical meridian of the retina on each side. This retinal area roughly corresponds to the reported strip of nasotemporal overlap, where both crossed and uncrossed retinofugal projections arise.
It is a classical concept of the central visual pathway organization of primates that all -anglion cells in the temporal retina project to the ipsilateral side of the brain, whereas those in the nasal retina project contralaterally, and that each side of the brain contains a representation of the contralateral visual hemifield3'3~. The data obtained from a number of non-primate mammalian species, however, suggest that part of the ipsilateral visual hemifield, as well as the contralateral hemifield, is represented in the superior colliculus (SC): the axons of a proportion of ganglion cells in the temporal retina have been indicated to terminate within the contralateral SC in the rat 7'!!'28, ground squirrel 4°, grey squirrel 18'22, cat1'2'1°'!2'i5'16'24'25'38 and tree shrew 18.22.The SC of the primates, such as bush baby 23 owl monkey 23, squirrel monkey ~9 and rhesus monkey 8'm°'39, has also been shown to receive input, not only from the nasal retina of the contralateral eye (input from the contralateral visual hemifield), but also
from the temporal retina of the contralateral eye (input from the ipsilateral one). It has been reported that there is a narrow strip of nasotemporal overlap in the projection from the vertical meridian of the retina in the cat 6'lt''2°'21'a2'aa'35, squirrel monkey 2t' and macaque monkeysS'ma'2t"37:this narrow strip of the retina, straddling the vertical merid. ian, project to both sides of the brain. No available data, however, have so far indicated whether crossed and uncrossed projections from such a retinal region originate from single ganglion cells, or distinct populations of ganglion cells each sending their axons exclusively to one side of the brain. The bifurcation of retinal ganglion cell axons to both sides of the brain has been shown in the rat ~'!7 (for review, see also ref. 14). In the present study, we therefore examined whether or not single retinal ganglion cells of the macaque monkey project bilaterally to the SC by way of axon collaterals.
Correspondence: N. Mizuno, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan.
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157 Ten Japanese monkeys (Macaca fuscata) of either sex weighing between 5.0-11.5 kg were used. The monkeys were tranquillized with an intramuscular injection of ketamine hydrochioride (10 m g / k g b.wt.), and then anesthetized with an intraperitoneal injection of sodium pentobarbital (30 m g / k g b.wt.), supplemented when necessary. They were positioned in a David-Kopf stereotaxic apparatus, and injected with Fast blue (FB, 5% aqueous suspension) into the right SC and with Diamidino yellow (DY, 3% aqueous suspension) into the left SC: the right SC was exposed by aspiration of the overlying parietal cortex, and a total volume of 1.0-2.0/zl of FB was injected into the SC under direct vision. On the left side, a total volume of 2.0-4.0 ~l of DY was injected stereotaxically into the SC region corresponding to the right SC region injected with FB; stereotaxic coordinates for the injection were determined with reference to the SC region on the right side. Each injection was made manually through a 10-/~l Hamilton microsyringe in a single or two needle penetration over 10 min, and the injection needle was kept in place for an additional 10 min before withdrawal. All surgical procedures were performed under aseptic conditions. After the operation, the monkeys were treated with antibiotics. The monkeys were allowed to survive for 7-14 days, re-anesthetized deeply, and then perfused transcardially with 0.1 M phosphate buffer (pH 7.4), followed by 10% formalin dissolved in the same buffer. After removal of the brains, brainstem blocks containing the injection sites in the SC were dissected out, saturated with 25% sucrose in the same buffer at 4°C, and then cut into serial frontal sections of 60-/zm thickness on a freezing microtome. The sections were mounted onto gelatin-coated glass slides. The retina was detached from the pigmented epithelium, flat-mounted onto a glass slide, and then coverslipped with a mixture of glycerol and water (1:1). The brain sections and retinas were observed with a Zeiss epifluorescence microscope. An ultraviolet filter providing excitation light of approximately 360 nm wavelength was used to view blue-emitting FB and yellow-emitting DY. After identification of the injection sites, the sections were lightly counterstained with 0.5% Neutral red.
In 6 monkeys, FB was injected into the rostral portion of the right SC, and DY was injected into the rostral portion of the left SC. In 4 of them, the injection site on each side was centered on the media', to intermediate part of the rostral SC. Out of these 4 monkeys, 1 monkey received large injections which covered not only the rostral portions of the bilateral SC, but also the olivary pretectal nucleus where retinal fibers have been known to terminate 27.29 (Fig. la). In the retina ipsilateral to the FB injection (contralateral to the DY injection), FB-positive and DY-positive cells were seen predominantly in its lower-temporal and lower-nasal region, respectively, whereas in the retina ipsilateral to the DY injection (contralateral to the FB injection), FB-positive and DY-positive cells were found mainly in its lower-nasal and lower-temporal region, respectively (Fig. la). The sizes of labeled cells were estimated by utilizing FB-positive cells; they comprised both large (more than 25/~m in diameter) and small (less than 25 /.tm in diameter) cells (Fig. 2). In each retina, about 30 cells were double-labeled with both FB and DY around the vertical meridian, a longitudinal axis through the fovea, where FB-positive and DY-positive cell populations overlapped with each other (Fig. la). These double-labeled cells were primarily of large type (Fig. 2), and were localized in the lower retina, with a higher density in the peripheral region distal to the fovea (Fig. la). In 3 monkeys, the injection site in the SC on each side was smaller, and was restricted to its rostral portion without tracer diffusion into the olivary pretectal nucleus (see Fig. lb as a representative monkey). After such bilateral SC injections, the distribution patterns of FB-positive and DY-positive cells in each retina were similar to those seen after the larger injections into the bilateral SC, although the number of labeled cells was smaller (compare Fig. lb with Fig. la). The double labeling of cells around the vertical meridian occurred in the lower retina on each side (Fig. lb); these double-labeled cells were also less numerous (about 10 cells per retina), and were composed of large type (Fig. 2). In 2 monkeys, the injection site on each side was centered on the lateral part of the SC (see Fig. lc as a
Fig. 1. Distribution of retrogradelylabeled ganglioncells in the retinae of 3 monkeys.Theywere injectedwith FB into the rostral SC on the right side (black areas) and with DY into the rostral SC on the left side (stippled areas), a: large injections which were centered on the medial to intermediate parts of the bilateral SC with diffusion into the olivary pretectal nucleus(OP). b: smaller injections which were centered on the medial to intermediate parts of the bilateral SC without diffusion into the OP. c: large injectionswhich were centered on the lateral parts of the bilateral SC. In each case, 3-4 frontal sections through the injectionsites are arranged rostrocaudally.The cells single labeled with FB or DY are indicated, respectively,with filled or open circles in a one-to-twentyratio, while the cells double labeled with both FB and DY are indicatedwith crosses in a one4o-four ratio. A star in each retina represents the fovea. IC, inferior colliculus;LL, lateral lemniscus;N, nasal: OD, optic disc: Pul, pulvinar; SCP, superior cerebellar peduncle: T, temporal; IV, trochlear nucleus.
158 representative monkey). In the retina ipsilateral to the FB injection (contralateral to the DY injection), the vast majority of FB-positive and DY-positive cells were found, respectively, in the upper-temporal and uppernasal region, and vice versa in the retina ipsilateral to the DY injection (contralateral to the FB injection) (Fig. lc); they were of large or small type (Fig. 2). In each retina, about 50 cells were double-labeled with both FB and DY. They were mostly large cells (Fig. 2), and were located around the vertical meridian of the upper retina, although they were seen more frequently in the peripheral region distal to the fovea (Fig. lc). In the remaining 4 monkeys, FB was injected into the medial (2 monkeys) or lateral (2 monkeys) part of the caudal SC on the right side, and DY was injected into the corresponding part of the left SC. In the retina ipsilateral to the FB injection (contralateral to the DY injection) into the medial part of the caudal SC (see Fig. 3a as a representative monkey), most of FB-positive and DY-positive cells were seen, respectively, in the lower-temporal and lower-nasal region, and vice
versa in the retina ipsilateral to the DY injection (contralateral to the FB injection) (Fig. 3a). On the other hand, in the retina ipsilateral to the FB injection (contralateral to the DY injection) into the lateral part of the caudal SC (see Fig. 3b as a representative monkey), most of the FB-positive and DY-positive cells were observed, respectively, in the upper-temporal and upper-nasal region, and vice versa in the retina ipsilateral to the DY injection (contralaterai to the FB injection) (Fig. 3b). In each retina of these monkeys, the area of distribution of FB-positive cells was totally separated from that of DY-positive cells: no overlap was seen (Fig. 3a,b), nor were double-labeled cells detected in the retina (Fig. 3a,b). The present study has provided morphological evidence that single retinal ganglion cells of the macaque monkey project to the bilateral SC by way of axon collaterals. Since such bilaterally projecting cells are primarily of large type (more than 25/~m in diameter), they are considered to belong to A cells of Leventhal et al. 27 or Pa cells of Perry et al. 3°, which appear to
Fig. 2. Photomicrographs of retrogradely labeled retinal ganglion cells in a monkey which were injected with FB into the rostral SC on the right side and with DY into the rostral SC on the left side. a,b: a cell double labeled with both FB and DY. c-e: cells labeled with FB and/or DY. Large cells double labeled with both FB and DY are specified by large arrows in c and dl large or small cells single labeled with FB are indicated, respectively, with large or small arrowheads in d and e; cells single labeled with DY are pointed out with small arrows in ¢-e. Bar = 25/.tin.
159 correspond to a cells (the morphological counterparts of Y cells) in the cat retina (for review, see also ref. 34). It has also been revealed in the present study that the ganglion cells sending axon collaterals to the bilateral SC are localized in a narrow strip around the vertical meridian. This retinal area appears to correspond to the reported strip of nasotemporal overlap, where both crossed and uncrossed retinofugal projections arise 5'!3'26'37. These bilaterally projecting cells have further been shown to be more abundant in the peripheral region distal to the fovea. This finding coincides well with previous reports 4'3°'36 that the number of A or Pa type of ganglion cells is increased with eccentricity from the fovea, particularly along the vertical meridian.
It has been known that the medio-lateral axis of the SC represents the lower-upper axis of the retina, while the rostro-caudal gradient of the SC represents the eccentricity from the fovea, especially from the vertical meridian (see refs. 8, 10, 23, 39). In good accordance with such reported retinotopy in the SC, the present results indicate that the rostral SC is the site for termination of retinotectal fibers originating from the nasotemporal overlap around the vertical meridian. In addition, it should be noted here that the pretectum may participate in bilateral projections of single retinal ganglion cells, because the A or Pa type of ganglion cells project not only to the SC but also to the pretectum 27'29. In fact, after bilateral injections into the rostral SC with diffusion into the olivary pretectal
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Fig. 3. Distribution of retrogradely labeled ganglion cells in the retinae of 2 monkeys. They were injected with FB into the caudal SC on the right side (black areas) and with DY injection into the caudal SC on the left side (stippled areas), a: injections which were centered on the medial parts of the bilateral SC. b: injections which were centered on the lateral parts of the bilateral SC. In each case, 2 frontal sections through the injection sites are arranged rostrocaudally. The cells single labeled with FB or DY are indicated, respectively, with filled or open circles in a one-to-twenty ratio. A star in each retina represents the fovea. Abbreviations are as in Fig. 1.
160 nucleus, double-labeled retinal ganglion cells were more numerous than those seen after bilateral SC injections, which were confined to the rostral SC (compare Fig. la with Fig. lb). Uncr,~ssed retinofugal projections are better developed in mammals with wider binocular visual fields, such as the cat, monkey and man, than in mammals with laterally situated eyes, such as the rat and rabbit TM. It is, therefore, conceivable that uncrossed retinofugal projections are phylogenetically newer than crossed ones. The bilateral projections of single retinal ganglion cells by way of axon collaterals may reflect the initial step towards the evolutionary acquisition of uncrossed projections. We are grateful for the photographic help of Mr. Akira Uesugi and the support of Drs. Ryosuke Fujimori, Satoru Fukuchi, Toshio Fukuda, Ritsu Hayashi, Sozaburo Hayashi, Mizuho Katsurada, Yutaka Kitani, Keiko Kumagai, Hiroshi Kuroda, Toshihiko Kuroda, Hiroshi Matsubara, Hiroshi Matsushima, Chisato Minakuchi, Masatoshi Nishio, Gonpei Niwa, Hajime Oda, Masahiko Ohbayashi, Sei-ichi Ohbayashi, Hiroyasu Ohtsuka, Shigeo Tamaki, Eizo Watanabe, Kazuo Yoshino and Toshiaki Yoshino. This work has been supported in part by Grants-in-Aid for Special Research on Priority Areas 04246106 and 02255107 and Scientific Research (B) 02454113 from the Ministry of Education, Science and Culture of Japan. ! Apter, J.T., Projection of the retina on superior colliculus of cats, J. Neurophysiol., 8 (1945) 123-134. 2 Berman, N. and Cynader, M., Comparison of receptive-field organization of the superior colliculus in Siamese and normal cats, J. Physiol., 224 (1972) 363-389. 3 Brouwer, B. and Zeeman, W.P.C., The projection of the retina in the primary optic neuron in monkeys, Brain, 49 (1926) !-35. 4 Bunt, A.H., Hendrickson, A.E., Land, J,S., Lund, R.D. and Fuchs, A.F., Monkey retinal ganglion cells: morphometric analysis and tracing of axonal projections, with a consideration of the peroxidase technique, J. Comp. Neurol., 164 (1975)265-286, 5 Bunt, A,H., Minckler, D.S. and Johanson, G.W,, Demonstration of bilateral projection of the central retina of the monkey with horseradish peroxidase neuronography, J. Comp. Neurol., 171 (1977) 619-630, 6 Cooper, M.L. and Pettigrew, J.D., The decussation of the retinothalamic pathway in the cat, with a note on the major meridians of the cat's eye, J. Comp. Neurol,, 187 (1979) 285-312. 7 Cowey, A. and Perry, V.H., The projection of the temporal retina in rats, studied by retrograde transport of horseradish peroxidase, .Exp. Brain Res., 35 (1979) 457-464. 8 Cowey, A. and Perry, V.H., The projection of the fovea to the superior colliculus in rhesus monkeys, Neuroscience, 5 (1980) 53-61, 9 Cunningham, T.J. and Freeman, J.A., Bilateral ganglion cell branches in the normal rat: a demonstration with electrophysiological collision and cobalt tracing methods, J. Comp. Neurol,, 172 (1977) 165-176. 10 Cynader, M. and Berman, N., Receptive-field organization of monkey superior colliculus, J. Neurophysiol., 35 (1972) 187-201. I1 Dreher, B., Sefton, A.J., Ni, S.Y.K. and Nisbett, G., The morphology, number, distribution and central projections of Class ! retinal ganglion ce[is in albino and hooded rats, Brain Behat,. Et'ol., 26 (1985) 10-48. 12 Feldon, S., Feldon. P. and Kruger, L., Topography of the retinal projection upon the superior colliculus of the cat, Vision Res., 10 (1970) ! 35- i 43. 13 Fukuda, Y., Sawai, H., Watanabe, M., Wakakuwa, K. and Mo-
rigiwa, K., Nasotemporal overlap of crossed and uncrossed retinal gar~glion cell projections in the Japanese monkey (Macaca ~stara), J. NeuroscL, 9 (1989) 2353-2373. 14 Gioili, R.A. and Towns, L.C., A review of axon collateralization in the mammalian visual system, Brain Behm'. Et'ol., 17 (1980) 364-390. 15 Hatting, J.K. and Guillery, R.W., Organization of retinocoilicular pathways in the cat, J. Comp. Neurol., 166 (1976) 133-144. 16 llling, R.-B. and W/issle, H., The retinal projection to the thalamus in the cat: a quantitative investigation and a comparison with the retinotectal pathway, J. Comp. Neurol., 202 (1981) 165-285. 17 Jeffery, G., Cowey, A. and Kuypers, H.G.J.M., Bifurcating retinal ganglion cell axons in the rat, demonstrated by retrograde double labelling, Exp. Brabz Res., 44 (1981) 34-40. 18 Kaas, J.H., Harting, J.K. and Guillery, R.W., Representation of the complete retina in the contralateral superior colliculus of some mammals, Brain Res., 65 (1974) 343-346. 19 Kadoya, S., Wolin, L.R. and Massopust, Jr., L.C., Photically evoked unit activity in the rectum opticum of the squirrel monkey, J. Comp. Neurol., 142 (1972) 495-508, 20 Kirk, D., Levick, W.R. and Cleland, B.G., The crossed or uncrossed destination of axons of sluggish-concentric and non-concentric cat retinal ganglion cells, with an overall synthesis of the visual field representation, Vision Res., 16 (1976) 233-236. 21 Kirk, D., Levick, W.R., Cleland, B.G. and Wiissle, H., Crossed and uncrossed representation of the visual field by brisk-sustained and brisk-transient cat retinal ganglion cells, Vision Res., 16 (1976) 225-231. 22 Lane, R.H., Allman, J.M. and Kaas, J.H., Representation of the visual field in the superior coiliculus of the grey squirrel (Sciums carolinensis) and the tree shrew (Tupaia glis), Brain Res., 26 (1971) 277-292, 23 Lane, R.H., Allman, J.M., Kaas, J.H, and Miezin, F.M., The visuotopic organization of the superior coUiculus of the owl monkey (Aotus trit'irgatus) and the bush baby (Galago senegalensis), Brain Res., 60 (1973) 335-349. 24 Lane, R.H., Kaas, J.H. and Allman, J.M., Visuotopic organization of the superior coiliculus in normal and Siamese cats, Brab~ Res., 70 (1974) 413-430. 25 Laties, A.M. and Sprague, J,M., The projection of optic fibers to the visual centers in the cat, Jr. Comp. Neurol., 127 (1966) 35-70. 26 Leventhal, A.G., Ault, S.J. and Vitek, D.J., The nasotemporal division in primate retina: the neural bases of macular sparing and splitting, Science, 240 (1988) 66-67, 27 Leventhal, A.G., Rodieck, R.W. and Dreher, B., Retinal ganglion cell classes in the Old World monkey: morphology and central projections, Science, 213 (1981) !139-1142. 28 Lund, R.D., Uncrossed visual pathways of hooded and albino rats, Science, 149 (1965) 1506-1507. 29 Perry, V.H. and Cowey, A., Retinal ganglion cells that project to the superior collicu|us and pretectum in tbe macaque monkey, Neuroscience, 12 (1984) I 125- I ! 37, 30 Perry, V.H,, Oehler, R. and Cowey, A., Retinal ganglion cells that project to the dorsal lateral geniculate nucleus in the macaque monkey, Neuroscience, 12 (1984) 1101-1123. 31 Polyak, S., The Vertebrate Visual System, University of Chicago Press, Chicago, 1957. 32 Sanderson, K.J. and Sherman, S,M., Nasotemporal overlap in visual field projected to lateral geniculate nucleus in the cat, J. Neurophysiol., 34 ( 1971) 453-466. 33 Stone, J., The naso-temporal division of the cat's retina, J. Comp. Neurol., 126 (1966) 585-600. 34 Stone, J. and Dreher, B., Parallel processing of information in the visual pathways: a general principle of sensory coding? Trends Neurosci., 5 (1982)441-446. 35 Stone, J. and Fukuda, Y., The naso-temporal division of the cat's retina re-examined in terms of Y-, X- and W-cells, J. Comp. Neurol., 155 (1974) 377-394. 36 Stone, J. and Johnston, E., The topography of primate retina: a study of the human, bushbaby and New- and Old-World monkeys, J. Comp, Neurol., 196 (1981) 205-223.
161 37 Stone, J., Leicester, J. and Sherman, S.M., The naso-temporal division of the monkey's retina, ./. Comp. Neurol., 150 (1973) 333-348 38 Straschill, M. and Hoffmann, K.P., Functional aspects of localization in the cat's rectum opticum, Brain Res., 13 (1969) 274-283.
39 Wilson, M.E. and Toyne, M.J., Retino-tectal and cortico-tectal projections in Macaca mulatta, Brabz Res., 24 (1970) 395-406. 40 Woolsey, C.N., Carlton, T.G., Kaas, J.H. and Earls, F.J., Projection of visual field on superior colliculus of ground squirrel (Citellus tridecemlineatus), Vision Res., 11 (1971) 115-127.
155
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25436
Single retinal ganglion cells sending axon collaterals to the bilateral superior colliculi: a fluorescent retrograde double-labeling study in the Japanese monkey ( Macaca fuscata) Yasuko Kondo
a, M a s a h i k o T a k a d a b, T e t s u r o K a y a h a r a c, Y u k i h i k o Yasui c, K a t s u m a N a k a n o c and Noboru Mizuno b
Departments of b Morphological Brain Science and a Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto (Japan) and c Department of Anatomy (1st Division), Mie Unicersity School of Medicine, Tsu, Mie (Japan) (Accepted 18 August 1992)
Key words: Visual system; Retinal ganglion ceil; Retina; Superior colliculus; Retinotectal projection; Bilateral projection; Axon collateralization; Monkey
Single retinal ganglion cells projecting bilaterally to the superior colliculi (SC) by way of axon collaterals were revealed in the Japanese monkey
(Macaca fuscata). After injecting Fast blue into the SC on one side and Diamidino yellow into the SC on the opposite side, some retinal ganglion cells were double-labeled with both tracers. Most of them were large cells (more than 25 ~m in diameter), and were localized in a narrow strip around the vertical meridian of the retina on each side. This retinal area roughly corresponds to the reported strip of nasotemporal overlap, where both crossed and uncrossed retinofugal projections arise.
It is a classical concept of the central visual pathway organization of primates that all -anglion cells in the temporal retina project to the ipsilateral side of the brain, whereas those in the nasal retina project contralaterally, and that each side of the brain contains a representation of the contralateral visual hemifield3'3~. The data obtained from a number of non-primate mammalian species, however, suggest that part of the ipsilateral visual hemifield, as well as the contralateral hemifield, is represented in the superior colliculus (SC): the axons of a proportion of ganglion cells in the temporal retina have been indicated to terminate within the contralateral SC in the rat 7'!!'28, ground squirrel 4°, grey squirrel 18'22, cat1'2'1°'!2'i5'16'24'25'38 and tree shrew 18.22.The SC of the primates, such as bush baby 23 owl monkey 23, squirrel monkey ~9 and rhesus monkey 8'm°'39, has also been shown to receive input, not only from the nasal retina of the contralateral eye (input from the contralateral visual hemifield), but also
from the temporal retina of the contralateral eye (input from the ipsilateral one). It has been reported that there is a narrow strip of nasotemporal overlap in the projection from the vertical meridian of the retina in the cat 6'lt''2°'21'a2'aa'35, squirrel monkey 2t' and macaque monkeysS'ma'2t"37:this narrow strip of the retina, straddling the vertical merid. ian, project to both sides of the brain. No available data, however, have so far indicated whether crossed and uncrossed projections from such a retinal region originate from single ganglion cells, or distinct populations of ganglion cells each sending their axons exclusively to one side of the brain. The bifurcation of retinal ganglion cell axons to both sides of the brain has been shown in the rat ~'!7 (for review, see also ref. 14). In the present study, we therefore examined whether or not single retinal ganglion cells of the macaque monkey project bilaterally to the SC by way of axon collaterals.
Correspondence: N. Mizuno, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan.
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157 Ten Japanese monkeys (Macaca fuscata) of either sex weighing between 5.0-11.5 kg were used. The monkeys were tranquillized with an intramuscular injection of ketamine hydrochioride (10 m g / k g b.wt.), and then anesthetized with an intraperitoneal injection of sodium pentobarbital (30 m g / k g b.wt.), supplemented when necessary. They were positioned in a David-Kopf stereotaxic apparatus, and injected with Fast blue (FB, 5% aqueous suspension) into the right SC and with Diamidino yellow (DY, 3% aqueous suspension) into the left SC: the right SC was exposed by aspiration of the overlying parietal cortex, and a total volume of 1.0-2.0/zl of FB was injected into the SC under direct vision. On the left side, a total volume of 2.0-4.0 ~l of DY was injected stereotaxically into the SC region corresponding to the right SC region injected with FB; stereotaxic coordinates for the injection were determined with reference to the SC region on the right side. Each injection was made manually through a 10-/~l Hamilton microsyringe in a single or two needle penetration over 10 min, and the injection needle was kept in place for an additional 10 min before withdrawal. All surgical procedures were performed under aseptic conditions. After the operation, the monkeys were treated with antibiotics. The monkeys were allowed to survive for 7-14 days, re-anesthetized deeply, and then perfused transcardially with 0.1 M phosphate buffer (pH 7.4), followed by 10% formalin dissolved in the same buffer. After removal of the brains, brainstem blocks containing the injection sites in the SC were dissected out, saturated with 25% sucrose in the same buffer at 4°C, and then cut into serial frontal sections of 60-/zm thickness on a freezing microtome. The sections were mounted onto gelatin-coated glass slides. The retina was detached from the pigmented epithelium, flat-mounted onto a glass slide, and then coverslipped with a mixture of glycerol and water (1:1). The brain sections and retinas were observed with a Zeiss epifluorescence microscope. An ultraviolet filter providing excitation light of approximately 360 nm wavelength was used to view blue-emitting FB and yellow-emitting DY. After identification of the injection sites, the sections were lightly counterstained with 0.5% Neutral red.
In 6 monkeys, FB was injected into the rostral portion of the right SC, and DY was injected into the rostral portion of the left SC. In 4 of them, the injection site on each side was centered on the media', to intermediate part of the rostral SC. Out of these 4 monkeys, 1 monkey received large injections which covered not only the rostral portions of the bilateral SC, but also the olivary pretectal nucleus where retinal fibers have been known to terminate 27.29 (Fig. la). In the retina ipsilateral to the FB injection (contralateral to the DY injection), FB-positive and DY-positive cells were seen predominantly in its lower-temporal and lower-nasal region, respectively, whereas in the retina ipsilateral to the DY injection (contralateral to the FB injection), FB-positive and DY-positive cells were found mainly in its lower-nasal and lower-temporal region, respectively (Fig. la). The sizes of labeled cells were estimated by utilizing FB-positive cells; they comprised both large (more than 25/~m in diameter) and small (less than 25 /.tm in diameter) cells (Fig. 2). In each retina, about 30 cells were double-labeled with both FB and DY around the vertical meridian, a longitudinal axis through the fovea, where FB-positive and DY-positive cell populations overlapped with each other (Fig. la). These double-labeled cells were primarily of large type (Fig. 2), and were localized in the lower retina, with a higher density in the peripheral region distal to the fovea (Fig. la). In 3 monkeys, the injection site in the SC on each side was smaller, and was restricted to its rostral portion without tracer diffusion into the olivary pretectal nucleus (see Fig. lb as a representative monkey). After such bilateral SC injections, the distribution patterns of FB-positive and DY-positive cells in each retina were similar to those seen after the larger injections into the bilateral SC, although the number of labeled cells was smaller (compare Fig. lb with Fig. la). The double labeling of cells around the vertical meridian occurred in the lower retina on each side (Fig. lb); these double-labeled cells were also less numerous (about 10 cells per retina), and were composed of large type (Fig. 2). In 2 monkeys, the injection site on each side was centered on the lateral part of the SC (see Fig. lc as a
Fig. 1. Distribution of retrogradelylabeled ganglioncells in the retinae of 3 monkeys.Theywere injectedwith FB into the rostral SC on the right side (black areas) and with DY into the rostral SC on the left side (stippled areas), a: large injections which were centered on the medial to intermediate parts of the bilateral SC with diffusion into the olivary pretectal nucleus(OP). b: smaller injections which were centered on the medial to intermediate parts of the bilateral SC without diffusion into the OP. c: large injectionswhich were centered on the lateral parts of the bilateral SC. In each case, 3-4 frontal sections through the injectionsites are arranged rostrocaudally.The cells single labeled with FB or DY are indicated, respectively,with filled or open circles in a one-to-twentyratio, while the cells double labeled with both FB and DY are indicatedwith crosses in a one4o-four ratio. A star in each retina represents the fovea. IC, inferior colliculus;LL, lateral lemniscus;N, nasal: OD, optic disc: Pul, pulvinar; SCP, superior cerebellar peduncle: T, temporal; IV, trochlear nucleus.
158 representative monkey). In the retina ipsilateral to the FB injection (contralateral to the DY injection), the vast majority of FB-positive and DY-positive cells were found, respectively, in the upper-temporal and uppernasal region, and vice versa in the retina ipsilateral to the DY injection (contralateral to the FB injection) (Fig. lc); they were of large or small type (Fig. 2). In each retina, about 50 cells were double-labeled with both FB and DY. They were mostly large cells (Fig. 2), and were located around the vertical meridian of the upper retina, although they were seen more frequently in the peripheral region distal to the fovea (Fig. lc). In the remaining 4 monkeys, FB was injected into the medial (2 monkeys) or lateral (2 monkeys) part of the caudal SC on the right side, and DY was injected into the corresponding part of the left SC. In the retina ipsilateral to the FB injection (contralateral to the DY injection) into the medial part of the caudal SC (see Fig. 3a as a representative monkey), most of FB-positive and DY-positive cells were seen, respectively, in the lower-temporal and lower-nasal region, and vice
versa in the retina ipsilateral to the DY injection (contralateral to the FB injection) (Fig. 3a). On the other hand, in the retina ipsilateral to the FB injection (contralateral to the DY injection) into the lateral part of the caudal SC (see Fig. 3b as a representative monkey), most of the FB-positive and DY-positive cells were observed, respectively, in the upper-temporal and upper-nasal region, and vice versa in the retina ipsilateral to the DY injection (contralaterai to the FB injection) (Fig. 3b). In each retina of these monkeys, the area of distribution of FB-positive cells was totally separated from that of DY-positive cells: no overlap was seen (Fig. 3a,b), nor were double-labeled cells detected in the retina (Fig. 3a,b). The present study has provided morphological evidence that single retinal ganglion cells of the macaque monkey project to the bilateral SC by way of axon collaterals. Since such bilaterally projecting cells are primarily of large type (more than 25/~m in diameter), they are considered to belong to A cells of Leventhal et al. 27 or Pa cells of Perry et al. 3°, which appear to
Fig. 2. Photomicrographs of retrogradely labeled retinal ganglion cells in a monkey which were injected with FB into the rostral SC on the right side and with DY into the rostral SC on the left side. a,b: a cell double labeled with both FB and DY. c-e: cells labeled with FB and/or DY. Large cells double labeled with both FB and DY are specified by large arrows in c and dl large or small cells single labeled with FB are indicated, respectively, with large or small arrowheads in d and e; cells single labeled with DY are pointed out with small arrows in ¢-e. Bar = 25/.tin.
159 correspond to a cells (the morphological counterparts of Y cells) in the cat retina (for review, see also ref. 34). It has also been revealed in the present study that the ganglion cells sending axon collaterals to the bilateral SC are localized in a narrow strip around the vertical meridian. This retinal area appears to correspond to the reported strip of nasotemporal overlap, where both crossed and uncrossed retinofugal projections arise 5'!3'26'37. These bilaterally projecting cells have further been shown to be more abundant in the peripheral region distal to the fovea. This finding coincides well with previous reports 4'3°'36 that the number of A or Pa type of ganglion cells is increased with eccentricity from the fovea, particularly along the vertical meridian.
It has been known that the medio-lateral axis of the SC represents the lower-upper axis of the retina, while the rostro-caudal gradient of the SC represents the eccentricity from the fovea, especially from the vertical meridian (see refs. 8, 10, 23, 39). In good accordance with such reported retinotopy in the SC, the present results indicate that the rostral SC is the site for termination of retinotectal fibers originating from the nasotemporal overlap around the vertical meridian. In addition, it should be noted here that the pretectum may participate in bilateral projections of single retinal ganglion cells, because the A or Pa type of ganglion cells project not only to the SC but also to the pretectum 27'29. In fact, after bilateral injections into the rostral SC with diffusion into the olivary pretectal
a
< '% . . . ,. .,.. , x . . . ' - "
.
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b
Fig. 3. Distribution of retrogradely labeled ganglion cells in the retinae of 2 monkeys. They were injected with FB into the caudal SC on the right side (black areas) and with DY injection into the caudal SC on the left side (stippled areas), a: injections which were centered on the medial parts of the bilateral SC. b: injections which were centered on the lateral parts of the bilateral SC. In each case, 2 frontal sections through the injection sites are arranged rostrocaudally. The cells single labeled with FB or DY are indicated, respectively, with filled or open circles in a one-to-twenty ratio. A star in each retina represents the fovea. Abbreviations are as in Fig. 1.
160 nucleus, double-labeled retinal ganglion cells were more numerous than those seen after bilateral SC injections, which were confined to the rostral SC (compare Fig. la with Fig. lb). Uncr,~ssed retinofugal projections are better developed in mammals with wider binocular visual fields, such as the cat, monkey and man, than in mammals with laterally situated eyes, such as the rat and rabbit TM. It is, therefore, conceivable that uncrossed retinofugal projections are phylogenetically newer than crossed ones. The bilateral projections of single retinal ganglion cells by way of axon collaterals may reflect the initial step towards the evolutionary acquisition of uncrossed projections. We are grateful for the photographic help of Mr. Akira Uesugi and the support of Drs. Ryosuke Fujimori, Satoru Fukuchi, Toshio Fukuda, Ritsu Hayashi, Sozaburo Hayashi, Mizuho Katsurada, Yutaka Kitani, Keiko Kumagai, Hiroshi Kuroda, Toshihiko Kuroda, Hiroshi Matsubara, Hiroshi Matsushima, Chisato Minakuchi, Masatoshi Nishio, Gonpei Niwa, Hajime Oda, Masahiko Ohbayashi, Sei-ichi Ohbayashi, Hiroyasu Ohtsuka, Shigeo Tamaki, Eizo Watanabe, Kazuo Yoshino and Toshiaki Yoshino. This work has been supported in part by Grants-in-Aid for Special Research on Priority Areas 04246106 and 02255107 and Scientific Research (B) 02454113 from the Ministry of Education, Science and Culture of Japan. ! Apter, J.T., Projection of the retina on superior colliculus of cats, J. Neurophysiol., 8 (1945) 123-134. 2 Berman, N. and Cynader, M., Comparison of receptive-field organization of the superior colliculus in Siamese and normal cats, J. Physiol., 224 (1972) 363-389. 3 Brouwer, B. and Zeeman, W.P.C., The projection of the retina in the primary optic neuron in monkeys, Brain, 49 (1926) !-35. 4 Bunt, A.H., Hendrickson, A.E., Land, J,S., Lund, R.D. and Fuchs, A.F., Monkey retinal ganglion cells: morphometric analysis and tracing of axonal projections, with a consideration of the peroxidase technique, J. Comp. Neurol., 164 (1975)265-286, 5 Bunt, A,H., Minckler, D.S. and Johanson, G.W,, Demonstration of bilateral projection of the central retina of the monkey with horseradish peroxidase neuronography, J. Comp. Neurol., 171 (1977) 619-630, 6 Cooper, M.L. and Pettigrew, J.D., The decussation of the retinothalamic pathway in the cat, with a note on the major meridians of the cat's eye, J. Comp. Neurol,, 187 (1979) 285-312. 7 Cowey, A. and Perry, V.H., The projection of the temporal retina in rats, studied by retrograde transport of horseradish peroxidase, .Exp. Brain Res., 35 (1979) 457-464. 8 Cowey, A. and Perry, V.H., The projection of the fovea to the superior colliculus in rhesus monkeys, Neuroscience, 5 (1980) 53-61, 9 Cunningham, T.J. and Freeman, J.A., Bilateral ganglion cell branches in the normal rat: a demonstration with electrophysiological collision and cobalt tracing methods, J. Comp. Neurol,, 172 (1977) 165-176. 10 Cynader, M. and Berman, N., Receptive-field organization of monkey superior colliculus, J. Neurophysiol., 35 (1972) 187-201. I1 Dreher, B., Sefton, A.J., Ni, S.Y.K. and Nisbett, G., The morphology, number, distribution and central projections of Class ! retinal ganglion ce[is in albino and hooded rats, Brain Behat,. Et'ol., 26 (1985) 10-48. 12 Feldon, S., Feldon. P. and Kruger, L., Topography of the retinal projection upon the superior colliculus of the cat, Vision Res., 10 (1970) ! 35- i 43. 13 Fukuda, Y., Sawai, H., Watanabe, M., Wakakuwa, K. and Mo-
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