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Autoradiographic demonstration of retinal projections to the brain stem structures in the rabbit using transneuronal tracing technique with special reference to the retinal projections to the inferior olive
EXPERIMENTAL
NEUROLOGY
78, 369-379 (1982)
Autoradiographic Demonstration of Retinal Projections to the Brain Stem Structures in the Rabbit Using Transneuronal Tracing Technique with Special Reference to the Retinal Projections to the Inferior Olive KUNIKO
UCHIDA,
NOBORU MIZUNO, TETSUO AND KAZUO ITOH’
SUGIMOTO,
Department of Anatomy (1st Division) and Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto, Japan Received May 3, 1982 Retinal projections to the brain stem structures in the rabbit were examined autoradiographically using transneuronal tracing technique. Three or four weeks after intraocular injections of tritiated proline and tritiated fucose, significant amounts of silver grains indicating transneuronal labeling of axon terminals were present bilaterally in the visual cortical areas, the dorsal portions of the medial geniculate nucleus, and the suprageniculate nucleus, and contralaterally in the thalamic reticular nucleus, the lateroposterior-pulvinar nuclear complex, the parabigeminal nucleus, the pontine tegmental reticular nucleus of Bechterew, the dorsolateral, lateral, and paramedian pontine nuclei, the pontine reticular formation, and the dorsal cap and beta nucleus of the inferior olive. The label in the pontine regions was probably due to the afferent fibers from the pretectal nuclei and the superior colliculus, and the label in the inferior olive was considered to depend on the uncrossed afferent fibers from the pretectal nuclei and the nuclei of the accessory optic tract.
INTRODUCTION Transneuronal transport of tritiated proline and tritiated fucose in the visual system has been observed in a variety of mammals that survived for ’ Requests for reprints should be sent to Dr. Noboru Mizuno, Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. Dr. Uchida is at the Department of Ophthalmology, Faculty of Medicine, Kyoto University. The authors acknowledge the photographic assistance of Mr. Akira Uesugi and the support of the Niwa Medical Research Foundation. 369 00 14-4886/82/ 110369-l 1$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
370
UCHIDA
ET AL.
rather long times after intraocular injection of large amounts of the radioactive isotopes [for reviews, see (7, 14, 31)]. The main purpose of those studies was to obtain the autoradiographic views of the retinocortical projections; the descriptions about transneuronal subcortical labeling were only tentative (14), or restricted to a particular nucleus (29). The present study attempted to demonstrate the secondary visual projections to the brain stem structures in the rabbit after intraocular injections of transneuronal tracers. METHODS Experiments were carried out in six adult albino rabbits weighing 2.5 to 3.0 kg. An equal mixture of tritiated L-fucose and tritiated L-proline (New England Nuclear) was injected into the vitreous body of one eye in each rabbit, which was anesthetized with pentobarbital sodium (30 mg/kg, iv. and 15 mg/kg, i.m.) and placed in a stereotaxic instrument. The injection in each rabbit was in two steps with a time interval of 1 week; 100 ~1 of the mixture (15 cLCi/pl) or 50 ~1 of the mixture (20 &i/pl) was injected in the first or second injection, respectively (total: 2.5 mCi in 150 ~1). After decreasing the intraocular pressure by puncturing the anterior chamber, pressure injections were made during a period of 1 h through a fine needle attached to a 0.25-ml Hamilton microsyringe that was mounted on a micromanipulator. After 2 or 3 weeks of postinjection survival, the rabbits were deeply anesthetized and perfused through the ascending aorta with 2 liters of 10% buffered Formalin. The brains were removed immediately and stored in 10% buffered Formalin. After saturation with a solution of 30% sucrose in 10% buffered Formalin, the brains were cut serially at 35 pm in the frontal plane on a freezing microtome and mounted onto gelatin-coated slides. These sections were defatted in xylene and coated with photosensitive emulsion (Kodak NTB-2, NTB-3, or Sakura NR-M2). After exposure for 4 to 6 weeks, the slides were developed with Kodak D-19, fixed, washed, and stained through the emulsion with cresyl violet. RESULTS After monocular injection of the radioactive isotopes a common, differential pattern of labeling was noted over certain regions in all six rabbits, although relatively high background labeling was present. All primary optic centers reported previously in the rabbit (8, 30, 32, 33) were heavily labeled in all rabbits (Fig. 1); dense label was seen bilaterally
RETINAL
PROJECTIONS
TO THE BRAIN STEM
371
FIG. 1. Line drawings of coronal sections through the rabbit forebrain and the midbrain, showing the pattern of label after injection of tritiated proline and tritiated fucose into the vitreous body of the left eye. Large or small black dots indicate heavy or light label, respectively. Abbreviations: CG-central gray, CP-cerebral peduncle, F-fomix, Flm-medial longitudinal fasciculus, HI-habenulointerpeduncular fasciculus, i-intermediate layer of the superior colliculus, IP-interpeduncular nucleus, LD-laterodorsal nucleus, LGd-dorsal lateral geniculate nucleus, LGv-ventral lateral geniculate nucleus, LP-PL-lateroposterior pulvinar nuclear complex, MD-mediodorsal nucleus, MC-medial geniculate nucleus, MT-mammillothalamic fasciculus, MTN-medial terminal nucleus of the accessory optic tract, NOolivary pretectal nucleus, NOT-nucleus of the optic tract, NPA-anterior pretectal nucleus, NPC-nucleus of the posterior commissure, NPP-posterior pretectal nucleus, OT-optic tract, p-deep layer of the superior colliculus, RN-red nucleus, S-super-h&l layer of the superior colliculus, SCH-suprachiasmatic nucleus, VB-ventrobasal nuclear complex.
372
UCHIDA
ET AL.
FIG. 2. Line drawings of coronal sections through the lower brain stem, showing the pattern of label after injection of the radioactive isotopes into the rabbit left eye. Large and small dots indicate heavy and light label, respectively. Abbreviations: III-oculomotor nucleus, IVtrochlear nucleus, BC-brachium conjunctivum, DB-decussation of the brachium conjunctivum, K-inferior colliculus, ML-medial lemniscus, NRT-pontine tegmental reticular nucleus of Bechterew, PBN-parabigeminal nucleus, PN-pontine nuclei, SC-superior COlliculus. For other abbreviations, see Fig. 1.
in the suprachiasmatic nucleus, and contralaterally in the dorsal and ventral lateral geniculate nuclei, nucleus of the optic tract, the olivary, anterior, and posterior pretectal nuclei, the superficial layer of the superior colliculus, the dorsal, lateral, and medial terminal nuclei of the accessory optic tract, and the lateral portions of the lateroposterior-pulvinar nuclear complex.
RETINAL
PROJECTIONS
TO THE
BRAIN
STEM
373
Dense label was also seen ipsilaterally in the medial portions of the dorsal geniculate nucleus. Light label was seen ipsilaterally in the dorsal portions of the ventral lateral geniculate nucleus, the dorsal portions of the posterior pretectal nucleus, the olivary pretectal nucleus, the nucleus of the optic tract, and the medial terminal nucleus of the optic tract (Fig. 1). The label in the superior colliculus ipsilateral to the injected eye was grouped into a few small patches in the rostrolateral portions of the colliculus (Figs. 1 and 2). In addition to the label within the known primary optic centers, light label was found in the visual cortex as well as in other secondary optic centers. In the striate cortical region contralateral to the injected eye, the labeling was seen in cortical layers II, III, IV, and VI; in other contralateral occipital regions and the ipsilateral striate region, the labeling was almost restricted to layer IV, as described by Hollander and Halbig (14). In subcortical regions, light label was seen bilaterally in the dorsal portions of the medial geniculate nucleus (Fig. l), and the suprageniculate nucleus (Fig. I), and contralaterally in the thalamic reticular nucleus (Fig. I), the parabigeminal nucleus (Figs. 2 and 3), the dorsolateral, lateral, and paramedian pontine nuclei (Figs. 2 and 3), the lateral portions of the pontine tegmental reticular nucleus of Bechterew (Fig. 2), the ventrolateral regions of the pontine tegmentum (Fig. 2), and the dorsal cap and beta nucleus of the inferior olive (Fig. 4). The label in the dorsal cap was seen in its whole rostrocaudal extent (Fig. 5). Light labeling of nerve fibers was noted in the medial longitudinal fasciculus and the predorsal fasciculus on the side contralateral to the injected eye, but not on the side ipsilateral to the injected eye. No labeled nerve cell bodies were found in the intermediate and deep layers of the superior colliculus. No significant label was noted in the cerebellum and the upper cervical cord segments. DISCUSSION In addition to the label in the well known retinal targets (8, 30, 32, 33), dense terminal label was observed in the lateral regions of the lateroposterior-pulvinar nuclear complex. Takahashi et al. (33) observed terminal label autoradiographically in the similar regions of the lateroposterior-pulvinar nuclear complex of a rabbit 24 h after intraocular injection of tritium leucine [cf. Fig. Id-f of (33)]. Therefore, at least some part of the label in the lateral regions of the posterolateral-pulvinar nuclear complex observed in our study might be due to direct retinopulvinar fibers, although the lateroposterior-pulvinar nuclear complex was reported to receive substantial amount of projection fibers from the superior colliculus and pretectum
374
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ET AL.
RETINAL
FIG.
nucleus
PROJECTIONS
4. Dark-field photomicrographs (B) in the right inferior olive.
showing X45.
TO THE
BRAIN
silver grains
375
STEM
in the dorsal
cap (DC)
and beta
in the rabbit (LO, 35), cat (1, 4, 6, 9, 11, 15, 20, 21), and monkey (3). In fact, recent studies using tracer techniques revealed the retinopulvinar fibers in the cat (5, 16, 19, 22) and macaque monkey (26). The light terminal label in the contralateral thalamic reticular nucleus resulted probably from transneuronal transport of the radioactive isotopes from the dorsal lateral geniculate nucleus [(29), also cf. (1 S)]. The parabigeminal nucleus, the suprageniculate nucleus, and the dorsal portions of the medial geniculate nucleus were most likely labeled transsynaptically via the superior colliculus [(35), also cf. (2, 12)]. In the pontine regions, the terminal label was observed in the pontine tegmental reticular nucleus of Bechterew, the paramedian, dorsolateral, and lateral pontine nuclei, and the pontine reticular formation. In the rabbit the uncrossed pretectopontine fibers have been described to terminate in the pontine tegmental reticular nucleus, the paramedian pontine nucleus, and the pontine reticular formation, and the uncrossed tectopontine fibers have been known to end in the dorsolateral and lateral pontine nuclei, and the pontine reticular formation [(27), also cf. (1, 4, 9, 15)]. The tectal fibers to FIG. 3. Dark-field photomicrographs (PBN, a) and the dorsolateral pontine cerebellar peduncle. X50.
showing silver grains in the parabigeminal nucleus nucleus (PN, b). CP-cerebral peduncle, MCP-middle
376
UCHIDA
ET AL.
FIG. 5. Line drawings of ,c’oss sections through the right inferior olive with dots depicting the pattern of label after injection of the radioactive isotopes in the contralateral eye. Abbreviations: B-beta nucleus, D-dorsal accessory olive, DC-dorsal cap, M-medial accessory olive, P-principal olive. Drawings 1 through 3 are the rostral-caudal extent of the label.
the dorsolateral pontine nucleus in the cat, however, were reported to arise mainly from the intermediate and deep layers of the superior colliculus (13), where no labeled nerve cell bodies were found in the present study. Therefore, the label in the dorsolateral and lateral pontine nuclei, at least some part of it, might be due to afferent fibers from the pretectal nuclei, although the possibilities of transsynaptic transport of the radioactive isotopes via neurons in the deep portions of the superficial layer of the superior colliculus could not be excluded [cf. (13)]. ‘The light terminal label in the dorsal cap and beta nucleus of the inferior olive contralateral to the injected eye was seen in the whole rostrocaudal extent of the regions. Maekawa and Simpson (23,24) reported in the rabbit that climbing fiber-responses evoked in the flocculus and nodulus by stimulating the optic nerve were mediated via the lateral portions of the pretectum, the central tegmental tract, and the rostral extent of the medial accessory olive. On the other hand, Mizuno et al. (27, 28) showed morphologically that fibers arising from the lateral portions of the pretectal regions in the rabbit descended ipsilaterally in the medial portions of the lower brain stem to end in the dorsal cap and beta nucleus of the inferior olive. Subsequently, these anatomic findings were confirmed and extended by Takeda and Maekawa (34), who showed in the rabbit that neurons in the nucleus of the optic tract as well as those in the dorsal and lateral terminal nuclei of the accessory optic tract were labeled ipsilaterally with horseradish peroxidase injected into the dorsal cap and beta nucleus. The existence of the uncrossed pretectoolivary fibers descending through the medial portions of the lower brain stem to end in the dorsal cap and beta nucleus was also reported in the cat (15, 37) and rat (36). Maekawa and Takeda (25) further reported that iontophoresis of horseradish peroxidase into the rostra1 portions of the dorsal cap labeled neurons ipsilaterally in the regions ventromedial to the red nucleus as well as in the regions between the substantia nigra and the medial lemniscus. According to their figures, most of these labeled neurons in the regions between the substantia nigra and the medial lemniscus appear to be situated within the
RETINAL
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STEM
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confines of the medial terminal nucleus of the accessory optic tract [cf. (17)]. The medial terminal nucleus of the accessory optic tract was heavily labeled contralaterally to the injected eye in our study. On the other hand, Maekawa and Takeda (25) assumed that the neurons in the ventromedial tegmental region of the midbrain receive optic signals from the ipsilateral eye that cross once at the optic chiasma to the contralateral pretectal region and then recross to the ipsilateral side through the posterior commissure, and that the neurons in the rostral half of the dorsal cap thus receive optic signals from the ipsilateral eye via the contralateral pretectal region, the posterior commissure, and the ipsilateral ventromedial tegmental region of the midbrain. In our study, however, the dense terminal label in the medial terminal nucleus of the accessory optic tract mainly resulted from the contralateral intraocular injection, and no significant label was found in the inferior olive ipsilateral to the injected eye. Therefore, if the transsynaptic label in the rostra1 half of the dorsal cap was due to afferent fibers from the medial terminal nucleus of the accessory optic tract, neurons in the rostral half of the dorsal cap must be considered to receive impulses in the optic fibers bisynaptically from the contralateral side. Thus, neurons in the rostra1 portions of the dorsal cap may receive impulses in the optic fibers bisynaptically from the contralateral side as well as trisynaptically from the ipsilateral side. REFERENCES 1. ALTMAN, J., AND M. B. CARPENTER. 1961. Fiber projections of the superior colliculus in the cat. J. Comp. Neural. 116: 157-117. 2. BALEYDIER, C., AND M. MAGNIN. 1979. Afferent and efferent connections of the parabigeminal nucleus in cat revealed by retrograde axonal transport of horseradish peroxidase. Bruin
Rex
161: 187-198.
3. BENEVENTO, L. A., AND J. H. FALLON. 1975. The ascending projections of the superior colhculus in the rhesus monkey (Mucaca mulatta). J. Camp. Neural. 160: 339-362. 4. BERMAN, N. 1977. Connections of the pretectum in the cat. J. Comp. Neurol. 174: 221254. 5. BERMAN, N., AND E. G. JONES. 1977. A retino-pulvinar projection in the cat. Brain Rex 134: 237-248. 6.
BERSON, D. M., AND A. M. GRAYBIEL. 1978. Parallel thalamic zones in the LP-pulvinar complex of the cat identified by their afferent and efferent connections. Brain Res. 147: 139-148.
7. DEBRUYN, E. J., AND V. A. CASAGRANDE. 1981. Demonstration of ocular dominance columns in a New World primate by means of monocular deprivation. Brain Rex 207: 453-458.
GIOLLI, R. A., AND M. D. DUTHRIE. 1969. The primary optic projections in the rabbit. An experimental degeneration study. .I. Comp. Neurol. 136: 99-126. 9. GRAHAM, J. 1977. An autoradiographic study of the efferent connections of the superior colhculus in the cat. J. Comp. Neurof. 173: 629-654. 10. GRAHAM, J., AND N. BERMAN. 198 1. Origin of the projections of the superior colliculus 8.
378
11. 12. 13.
14.
15. 16.
17.
18. 19. 20.
21. 22.
23.
24. 25.
26.
27. 28.
29.
UCHIDA
ET AL.
to the dorsal geniculate nucleus and the pulvinar in the rabbit. Neurosci. Left. 26: lOl106. GRAYBIEL, A. M. 1972. Some extrageniculate visual pathways in the cat. Invest. Ophthdmol. 11: 322-332. GFCAYBIEL, A. M. 1978. A satellite system of the superior colliculus: the parabigeminal nucleus and its projections to the superficial colliculus layers. Bruin Res. 145: 365-374. HASHIKAWA, T., AND K. KAWAMURA. 1977. Identification of cells of origin of tectopontine fibers in the cat superior colliculus: an experimental study with the horseradish peroxidase method. Brain Res. 130: 65-79. HOLL~NDER, H., AND W. HALBIG. 1980. Topography of retinal representation in the rabbit cortex: an experimental study using transneuronal and retrograde tracing techniques. J. Comp. Neurol. 193: 701-7 10. ITOH, K. 1977. Efferent projections of the pretectum in the cat. Exp. Brain Res. 30: 89105. ITOH, K., N. MIZUNO, T. SUGIMOTO, S. NOMURA, Y. NAKAMURA, AND A. KONISHI. 1979. A cerebella-pulvino-cortical and a retino-pulvino-cortical pathways in the cat as revealed by the use of the anterograde and retrograde transport of horseradish peroxidase. J. Comp. Neurol. 187: 349-358. IWAHORI, N., K. UCHIDA, AND N. MIZUNO. 198 1. A Golgi analysis of the accessory optic fibers terminating in the medial terminal nucleus of the mouse accessory optic system. Neurosci. Lett. 23: 229-232. JONES, E. G. 1975. Some aspects of the organization of the thalamic reticular complex. J. Comp. Neurol. 162: 285-308. KAWAMURA, S., N. FUKUSHIMA, AND S. HATTORI. 1979. Topographical origin and ganglion cell type of the retino-pulvinar projection in the cat. Brain Res. 173: 419-429. KAWAMURA, S., N. FUKUSHIMA, S. HATTORI, AND M. KUDO. 1980. Laminar segregation of cells of origin of ascending projections from the superficial layers of the superior colliculus in the cat. Bruin Res. 184: 486-490. KAWAMURA, S., AND E. KOBAYASHI. 1975. Identification of laminar origin of some tectothalamic fibers in the cat. Brain Res. 91: 281-285. LEVENTHAL, A. G., J. KEENS, AND I. TURK. 1980. The afferent ganglion cells and cortical projections of the retinal recipient zone (RRZ) of the cat’s “pulvinar complex.” J. Comp. Neurol. 194: 535-554. MAEKAWA, K., AND J. I. SIMPSON. 1972. Climbing fiber activation of Purkinje cells in the flocculus by impulses transferred through the visual pathway. Brain Res. 39: 245251. MAEKAWA, K., AND J. I. SIMPSON. 1973. Climbing fiber responses evoked in vestibulocerebellum of rabbit from visual system. J. Neurophysiol. 36: 649-666. MAEKAWA, K., AND T. TAKEDA. 1979. Origin of descending afferents to the rostral part of dorsal cap of inferior olive which transfers contralateral optic activities to the flocculus. A horseradish peroxidase study. Brain Res. 172: 393-405. MIZUNO, N., K. ITOH, K. UCHIDA, M. UEMURA-SUMI, AND R. MATSUSHIMA. 1982. A retino-pulvinar projection in the macaque monkey as visualized by the use of anterograde transport of horseradish peroxidase. Neurosci. Lett. 30: 199-203. MIZUNO, N., K. MOCHIZUKI, C. AKIMOTO, AND R. MATSUSHIMA. 1973. Pretectal projections to the inferior olive in the rabbit. Exp. Neurol. 39: 498-506. MIZUNO, N., Y. NAKAMURA, AND N. IWAHORI. 1974. An electron microscope study of the dorsal cap of the inferior olive in the rabbit, with special reference to the pretectoolivary fibers. Brain Res. 77: 385-395. MONTERO, V. M., R. W. GUILLERY, AND C. N. WOOLSEY. 1977. Retinotopic organization
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within the thalamic reticular nucleus demonstrated by a double label autoradiographic technique. Brain Res. 138: Ml-42 1. 30. SANDERSON, K. J. 1975. Retinogeniculate projections in the rabbits of the albino allelomorphic series. J. Comp. Neurol. 159: 15-28. 31. SANDERSON, K. J., J. R. HAIGHT, AND L. J. PEARSON. 1980. Transneuronal transport of tritiated fucose and proline in the visual pathways of the brushtailed possum, Trichosurus vulpecula.
Neurosci.
Lett.
32. SCALIA, F. 1972. The termination J. Camp.
Neural.
20: 243-248.
of retinal axons in the pretectal region of mammals.
145: 223-258.
33. TAKAHASHI, E. S., T. L. HICKEY, AND C. W. OYSTER. 1977. Retinogeniculate projections in the rabbit: an autoradiographic study. J. Comp. Neural. 175: I- 12. 34. TAKEDA, T., AND K. MAEKAWA. 1976. The origin of the pretecto-olivary tract. A study using the horseradish peroxidase method. Brain Res. 117: 319-325. 35. TARLOV, E. C., AND R. Y. MOORE. 1966. The tectothalamic connections in the brain of the rabbit. J. Camp. Neurol. 126: 403-422. 36. TERASAWA, K., K. OTANI, AND J. YAMADA. 1979. Descending pathways of the nucleus of the optic tract in the rat. Brain Res. 173: 405-417. 37. WALBERG, F., T. NORDBY, K. P. HOFFMANN, AND H. HOLL~NDER. 1981. Olivary afferents from the pretectal nuclei in the cat. Anat. Embryol. 161: 291-304.
NEUROLOGY
78, 369-379 (1982)
Autoradiographic Demonstration of Retinal Projections to the Brain Stem Structures in the Rabbit Using Transneuronal Tracing Technique with Special Reference to the Retinal Projections to the Inferior Olive KUNIKO
UCHIDA,
NOBORU MIZUNO, TETSUO AND KAZUO ITOH’
SUGIMOTO,
Department of Anatomy (1st Division) and Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto, Japan Received May 3, 1982 Retinal projections to the brain stem structures in the rabbit were examined autoradiographically using transneuronal tracing technique. Three or four weeks after intraocular injections of tritiated proline and tritiated fucose, significant amounts of silver grains indicating transneuronal labeling of axon terminals were present bilaterally in the visual cortical areas, the dorsal portions of the medial geniculate nucleus, and the suprageniculate nucleus, and contralaterally in the thalamic reticular nucleus, the lateroposterior-pulvinar nuclear complex, the parabigeminal nucleus, the pontine tegmental reticular nucleus of Bechterew, the dorsolateral, lateral, and paramedian pontine nuclei, the pontine reticular formation, and the dorsal cap and beta nucleus of the inferior olive. The label in the pontine regions was probably due to the afferent fibers from the pretectal nuclei and the superior colliculus, and the label in the inferior olive was considered to depend on the uncrossed afferent fibers from the pretectal nuclei and the nuclei of the accessory optic tract.
INTRODUCTION Transneuronal transport of tritiated proline and tritiated fucose in the visual system has been observed in a variety of mammals that survived for ’ Requests for reprints should be sent to Dr. Noboru Mizuno, Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. Dr. Uchida is at the Department of Ophthalmology, Faculty of Medicine, Kyoto University. The authors acknowledge the photographic assistance of Mr. Akira Uesugi and the support of the Niwa Medical Research Foundation. 369 00 14-4886/82/ 110369-l 1$02.00/O Copyright All rights
0 1982 by Academic Press. Inc. of reproduction in any form reserved.
370
UCHIDA
ET AL.
rather long times after intraocular injection of large amounts of the radioactive isotopes [for reviews, see (7, 14, 31)]. The main purpose of those studies was to obtain the autoradiographic views of the retinocortical projections; the descriptions about transneuronal subcortical labeling were only tentative (14), or restricted to a particular nucleus (29). The present study attempted to demonstrate the secondary visual projections to the brain stem structures in the rabbit after intraocular injections of transneuronal tracers. METHODS Experiments were carried out in six adult albino rabbits weighing 2.5 to 3.0 kg. An equal mixture of tritiated L-fucose and tritiated L-proline (New England Nuclear) was injected into the vitreous body of one eye in each rabbit, which was anesthetized with pentobarbital sodium (30 mg/kg, iv. and 15 mg/kg, i.m.) and placed in a stereotaxic instrument. The injection in each rabbit was in two steps with a time interval of 1 week; 100 ~1 of the mixture (15 cLCi/pl) or 50 ~1 of the mixture (20 &i/pl) was injected in the first or second injection, respectively (total: 2.5 mCi in 150 ~1). After decreasing the intraocular pressure by puncturing the anterior chamber, pressure injections were made during a period of 1 h through a fine needle attached to a 0.25-ml Hamilton microsyringe that was mounted on a micromanipulator. After 2 or 3 weeks of postinjection survival, the rabbits were deeply anesthetized and perfused through the ascending aorta with 2 liters of 10% buffered Formalin. The brains were removed immediately and stored in 10% buffered Formalin. After saturation with a solution of 30% sucrose in 10% buffered Formalin, the brains were cut serially at 35 pm in the frontal plane on a freezing microtome and mounted onto gelatin-coated slides. These sections were defatted in xylene and coated with photosensitive emulsion (Kodak NTB-2, NTB-3, or Sakura NR-M2). After exposure for 4 to 6 weeks, the slides were developed with Kodak D-19, fixed, washed, and stained through the emulsion with cresyl violet. RESULTS After monocular injection of the radioactive isotopes a common, differential pattern of labeling was noted over certain regions in all six rabbits, although relatively high background labeling was present. All primary optic centers reported previously in the rabbit (8, 30, 32, 33) were heavily labeled in all rabbits (Fig. 1); dense label was seen bilaterally
RETINAL
PROJECTIONS
TO THE BRAIN STEM
371
FIG. 1. Line drawings of coronal sections through the rabbit forebrain and the midbrain, showing the pattern of label after injection of tritiated proline and tritiated fucose into the vitreous body of the left eye. Large or small black dots indicate heavy or light label, respectively. Abbreviations: CG-central gray, CP-cerebral peduncle, F-fomix, Flm-medial longitudinal fasciculus, HI-habenulointerpeduncular fasciculus, i-intermediate layer of the superior colliculus, IP-interpeduncular nucleus, LD-laterodorsal nucleus, LGd-dorsal lateral geniculate nucleus, LGv-ventral lateral geniculate nucleus, LP-PL-lateroposterior pulvinar nuclear complex, MD-mediodorsal nucleus, MC-medial geniculate nucleus, MT-mammillothalamic fasciculus, MTN-medial terminal nucleus of the accessory optic tract, NOolivary pretectal nucleus, NOT-nucleus of the optic tract, NPA-anterior pretectal nucleus, NPC-nucleus of the posterior commissure, NPP-posterior pretectal nucleus, OT-optic tract, p-deep layer of the superior colliculus, RN-red nucleus, S-super-h&l layer of the superior colliculus, SCH-suprachiasmatic nucleus, VB-ventrobasal nuclear complex.
372
UCHIDA
ET AL.
FIG. 2. Line drawings of coronal sections through the lower brain stem, showing the pattern of label after injection of the radioactive isotopes into the rabbit left eye. Large and small dots indicate heavy and light label, respectively. Abbreviations: III-oculomotor nucleus, IVtrochlear nucleus, BC-brachium conjunctivum, DB-decussation of the brachium conjunctivum, K-inferior colliculus, ML-medial lemniscus, NRT-pontine tegmental reticular nucleus of Bechterew, PBN-parabigeminal nucleus, PN-pontine nuclei, SC-superior COlliculus. For other abbreviations, see Fig. 1.
in the suprachiasmatic nucleus, and contralaterally in the dorsal and ventral lateral geniculate nuclei, nucleus of the optic tract, the olivary, anterior, and posterior pretectal nuclei, the superficial layer of the superior colliculus, the dorsal, lateral, and medial terminal nuclei of the accessory optic tract, and the lateral portions of the lateroposterior-pulvinar nuclear complex.
RETINAL
PROJECTIONS
TO THE
BRAIN
STEM
373
Dense label was also seen ipsilaterally in the medial portions of the dorsal geniculate nucleus. Light label was seen ipsilaterally in the dorsal portions of the ventral lateral geniculate nucleus, the dorsal portions of the posterior pretectal nucleus, the olivary pretectal nucleus, the nucleus of the optic tract, and the medial terminal nucleus of the optic tract (Fig. 1). The label in the superior colliculus ipsilateral to the injected eye was grouped into a few small patches in the rostrolateral portions of the colliculus (Figs. 1 and 2). In addition to the label within the known primary optic centers, light label was found in the visual cortex as well as in other secondary optic centers. In the striate cortical region contralateral to the injected eye, the labeling was seen in cortical layers II, III, IV, and VI; in other contralateral occipital regions and the ipsilateral striate region, the labeling was almost restricted to layer IV, as described by Hollander and Halbig (14). In subcortical regions, light label was seen bilaterally in the dorsal portions of the medial geniculate nucleus (Fig. l), and the suprageniculate nucleus (Fig. I), and contralaterally in the thalamic reticular nucleus (Fig. I), the parabigeminal nucleus (Figs. 2 and 3), the dorsolateral, lateral, and paramedian pontine nuclei (Figs. 2 and 3), the lateral portions of the pontine tegmental reticular nucleus of Bechterew (Fig. 2), the ventrolateral regions of the pontine tegmentum (Fig. 2), and the dorsal cap and beta nucleus of the inferior olive (Fig. 4). The label in the dorsal cap was seen in its whole rostrocaudal extent (Fig. 5). Light labeling of nerve fibers was noted in the medial longitudinal fasciculus and the predorsal fasciculus on the side contralateral to the injected eye, but not on the side ipsilateral to the injected eye. No labeled nerve cell bodies were found in the intermediate and deep layers of the superior colliculus. No significant label was noted in the cerebellum and the upper cervical cord segments. DISCUSSION In addition to the label in the well known retinal targets (8, 30, 32, 33), dense terminal label was observed in the lateral regions of the lateroposterior-pulvinar nuclear complex. Takahashi et al. (33) observed terminal label autoradiographically in the similar regions of the lateroposterior-pulvinar nuclear complex of a rabbit 24 h after intraocular injection of tritium leucine [cf. Fig. Id-f of (33)]. Therefore, at least some part of the label in the lateral regions of the posterolateral-pulvinar nuclear complex observed in our study might be due to direct retinopulvinar fibers, although the lateroposterior-pulvinar nuclear complex was reported to receive substantial amount of projection fibers from the superior colliculus and pretectum
374
UCHIDA
ET AL.
RETINAL
FIG.
nucleus
PROJECTIONS
4. Dark-field photomicrographs (B) in the right inferior olive.
showing X45.
TO THE
BRAIN
silver grains
375
STEM
in the dorsal
cap (DC)
and beta
in the rabbit (LO, 35), cat (1, 4, 6, 9, 11, 15, 20, 21), and monkey (3). In fact, recent studies using tracer techniques revealed the retinopulvinar fibers in the cat (5, 16, 19, 22) and macaque monkey (26). The light terminal label in the contralateral thalamic reticular nucleus resulted probably from transneuronal transport of the radioactive isotopes from the dorsal lateral geniculate nucleus [(29), also cf. (1 S)]. The parabigeminal nucleus, the suprageniculate nucleus, and the dorsal portions of the medial geniculate nucleus were most likely labeled transsynaptically via the superior colliculus [(35), also cf. (2, 12)]. In the pontine regions, the terminal label was observed in the pontine tegmental reticular nucleus of Bechterew, the paramedian, dorsolateral, and lateral pontine nuclei, and the pontine reticular formation. In the rabbit the uncrossed pretectopontine fibers have been described to terminate in the pontine tegmental reticular nucleus, the paramedian pontine nucleus, and the pontine reticular formation, and the uncrossed tectopontine fibers have been known to end in the dorsolateral and lateral pontine nuclei, and the pontine reticular formation [(27), also cf. (1, 4, 9, 15)]. The tectal fibers to FIG. 3. Dark-field photomicrographs (PBN, a) and the dorsolateral pontine cerebellar peduncle. X50.
showing silver grains in the parabigeminal nucleus nucleus (PN, b). CP-cerebral peduncle, MCP-middle
376
UCHIDA
ET AL.
FIG. 5. Line drawings of ,c’oss sections through the right inferior olive with dots depicting the pattern of label after injection of the radioactive isotopes in the contralateral eye. Abbreviations: B-beta nucleus, D-dorsal accessory olive, DC-dorsal cap, M-medial accessory olive, P-principal olive. Drawings 1 through 3 are the rostral-caudal extent of the label.
the dorsolateral pontine nucleus in the cat, however, were reported to arise mainly from the intermediate and deep layers of the superior colliculus (13), where no labeled nerve cell bodies were found in the present study. Therefore, the label in the dorsolateral and lateral pontine nuclei, at least some part of it, might be due to afferent fibers from the pretectal nuclei, although the possibilities of transsynaptic transport of the radioactive isotopes via neurons in the deep portions of the superficial layer of the superior colliculus could not be excluded [cf. (13)]. ‘The light terminal label in the dorsal cap and beta nucleus of the inferior olive contralateral to the injected eye was seen in the whole rostrocaudal extent of the regions. Maekawa and Simpson (23,24) reported in the rabbit that climbing fiber-responses evoked in the flocculus and nodulus by stimulating the optic nerve were mediated via the lateral portions of the pretectum, the central tegmental tract, and the rostral extent of the medial accessory olive. On the other hand, Mizuno et al. (27, 28) showed morphologically that fibers arising from the lateral portions of the pretectal regions in the rabbit descended ipsilaterally in the medial portions of the lower brain stem to end in the dorsal cap and beta nucleus of the inferior olive. Subsequently, these anatomic findings were confirmed and extended by Takeda and Maekawa (34), who showed in the rabbit that neurons in the nucleus of the optic tract as well as those in the dorsal and lateral terminal nuclei of the accessory optic tract were labeled ipsilaterally with horseradish peroxidase injected into the dorsal cap and beta nucleus. The existence of the uncrossed pretectoolivary fibers descending through the medial portions of the lower brain stem to end in the dorsal cap and beta nucleus was also reported in the cat (15, 37) and rat (36). Maekawa and Takeda (25) further reported that iontophoresis of horseradish peroxidase into the rostra1 portions of the dorsal cap labeled neurons ipsilaterally in the regions ventromedial to the red nucleus as well as in the regions between the substantia nigra and the medial lemniscus. According to their figures, most of these labeled neurons in the regions between the substantia nigra and the medial lemniscus appear to be situated within the
RETINAL
PROJECTIONS
TO THE
BRAIN
STEM
377
confines of the medial terminal nucleus of the accessory optic tract [cf. (17)]. The medial terminal nucleus of the accessory optic tract was heavily labeled contralaterally to the injected eye in our study. On the other hand, Maekawa and Takeda (25) assumed that the neurons in the ventromedial tegmental region of the midbrain receive optic signals from the ipsilateral eye that cross once at the optic chiasma to the contralateral pretectal region and then recross to the ipsilateral side through the posterior commissure, and that the neurons in the rostral half of the dorsal cap thus receive optic signals from the ipsilateral eye via the contralateral pretectal region, the posterior commissure, and the ipsilateral ventromedial tegmental region of the midbrain. In our study, however, the dense terminal label in the medial terminal nucleus of the accessory optic tract mainly resulted from the contralateral intraocular injection, and no significant label was found in the inferior olive ipsilateral to the injected eye. Therefore, if the transsynaptic label in the rostra1 half of the dorsal cap was due to afferent fibers from the medial terminal nucleus of the accessory optic tract, neurons in the rostral half of the dorsal cap must be considered to receive impulses in the optic fibers bisynaptically from the contralateral side. Thus, neurons in the rostra1 portions of the dorsal cap may receive impulses in the optic fibers bisynaptically from the contralateral side as well as trisynaptically from the ipsilateral side. REFERENCES 1. ALTMAN, J., AND M. B. CARPENTER. 1961. Fiber projections of the superior colliculus in the cat. J. Comp. Neural. 116: 157-117. 2. BALEYDIER, C., AND M. MAGNIN. 1979. Afferent and efferent connections of the parabigeminal nucleus in cat revealed by retrograde axonal transport of horseradish peroxidase. Bruin
Rex
161: 187-198.
3. BENEVENTO, L. A., AND J. H. FALLON. 1975. The ascending projections of the superior colhculus in the rhesus monkey (Mucaca mulatta). J. Camp. Neural. 160: 339-362. 4. BERMAN, N. 1977. Connections of the pretectum in the cat. J. Comp. Neurol. 174: 221254. 5. BERMAN, N., AND E. G. JONES. 1977. A retino-pulvinar projection in the cat. Brain Rex 134: 237-248. 6.
BERSON, D. M., AND A. M. GRAYBIEL. 1978. Parallel thalamic zones in the LP-pulvinar complex of the cat identified by their afferent and efferent connections. Brain Res. 147: 139-148.
7. DEBRUYN, E. J., AND V. A. CASAGRANDE. 1981. Demonstration of ocular dominance columns in a New World primate by means of monocular deprivation. Brain Rex 207: 453-458.
GIOLLI, R. A., AND M. D. DUTHRIE. 1969. The primary optic projections in the rabbit. An experimental degeneration study. .I. Comp. Neurol. 136: 99-126. 9. GRAHAM, J. 1977. An autoradiographic study of the efferent connections of the superior colhculus in the cat. J. Comp. Neurof. 173: 629-654. 10. GRAHAM, J., AND N. BERMAN. 198 1. Origin of the projections of the superior colliculus 8.
378
11. 12. 13.
14.
15. 16.
17.
18. 19. 20.
21. 22.
23.
24. 25.
26.
27. 28.
29.
UCHIDA
ET AL.
to the dorsal geniculate nucleus and the pulvinar in the rabbit. Neurosci. Left. 26: lOl106. GRAYBIEL, A. M. 1972. Some extrageniculate visual pathways in the cat. Invest. Ophthdmol. 11: 322-332. GFCAYBIEL, A. M. 1978. A satellite system of the superior colliculus: the parabigeminal nucleus and its projections to the superficial colliculus layers. Bruin Res. 145: 365-374. HASHIKAWA, T., AND K. KAWAMURA. 1977. Identification of cells of origin of tectopontine fibers in the cat superior colliculus: an experimental study with the horseradish peroxidase method. Brain Res. 130: 65-79. HOLL~NDER, H., AND W. HALBIG. 1980. Topography of retinal representation in the rabbit cortex: an experimental study using transneuronal and retrograde tracing techniques. J. Comp. Neurol. 193: 701-7 10. ITOH, K. 1977. Efferent projections of the pretectum in the cat. Exp. Brain Res. 30: 89105. ITOH, K., N. MIZUNO, T. SUGIMOTO, S. NOMURA, Y. NAKAMURA, AND A. KONISHI. 1979. A cerebella-pulvino-cortical and a retino-pulvino-cortical pathways in the cat as revealed by the use of the anterograde and retrograde transport of horseradish peroxidase. J. Comp. Neurol. 187: 349-358. IWAHORI, N., K. UCHIDA, AND N. MIZUNO. 198 1. A Golgi analysis of the accessory optic fibers terminating in the medial terminal nucleus of the mouse accessory optic system. Neurosci. Lett. 23: 229-232. JONES, E. G. 1975. Some aspects of the organization of the thalamic reticular complex. J. Comp. Neurol. 162: 285-308. KAWAMURA, S., N. FUKUSHIMA, AND S. HATTORI. 1979. Topographical origin and ganglion cell type of the retino-pulvinar projection in the cat. Brain Res. 173: 419-429. KAWAMURA, S., N. FUKUSHIMA, S. HATTORI, AND M. KUDO. 1980. Laminar segregation of cells of origin of ascending projections from the superficial layers of the superior colliculus in the cat. Bruin Res. 184: 486-490. KAWAMURA, S., AND E. KOBAYASHI. 1975. Identification of laminar origin of some tectothalamic fibers in the cat. Brain Res. 91: 281-285. LEVENTHAL, A. G., J. KEENS, AND I. TURK. 1980. The afferent ganglion cells and cortical projections of the retinal recipient zone (RRZ) of the cat’s “pulvinar complex.” J. Comp. Neurol. 194: 535-554. MAEKAWA, K., AND J. I. SIMPSON. 1972. Climbing fiber activation of Purkinje cells in the flocculus by impulses transferred through the visual pathway. Brain Res. 39: 245251. MAEKAWA, K., AND J. I. SIMPSON. 1973. Climbing fiber responses evoked in vestibulocerebellum of rabbit from visual system. J. Neurophysiol. 36: 649-666. MAEKAWA, K., AND T. TAKEDA. 1979. Origin of descending afferents to the rostral part of dorsal cap of inferior olive which transfers contralateral optic activities to the flocculus. A horseradish peroxidase study. Brain Res. 172: 393-405. MIZUNO, N., K. ITOH, K. UCHIDA, M. UEMURA-SUMI, AND R. MATSUSHIMA. 1982. A retino-pulvinar projection in the macaque monkey as visualized by the use of anterograde transport of horseradish peroxidase. Neurosci. Lett. 30: 199-203. MIZUNO, N., K. MOCHIZUKI, C. AKIMOTO, AND R. MATSUSHIMA. 1973. Pretectal projections to the inferior olive in the rabbit. Exp. Neurol. 39: 498-506. MIZUNO, N., Y. NAKAMURA, AND N. IWAHORI. 1974. An electron microscope study of the dorsal cap of the inferior olive in the rabbit, with special reference to the pretectoolivary fibers. Brain Res. 77: 385-395. MONTERO, V. M., R. W. GUILLERY, AND C. N. WOOLSEY. 1977. Retinotopic organization
RETINAL
PROJECTIONS
TO THE BRAIN STEM
379
within the thalamic reticular nucleus demonstrated by a double label autoradiographic technique. Brain Res. 138: Ml-42 1. 30. SANDERSON, K. J. 1975. Retinogeniculate projections in the rabbits of the albino allelomorphic series. J. Comp. Neurol. 159: 15-28. 31. SANDERSON, K. J., J. R. HAIGHT, AND L. J. PEARSON. 1980. Transneuronal transport of tritiated fucose and proline in the visual pathways of the brushtailed possum, Trichosurus vulpecula.
Neurosci.
Lett.
32. SCALIA, F. 1972. The termination J. Camp.
Neural.
20: 243-248.
of retinal axons in the pretectal region of mammals.
145: 223-258.
33. TAKAHASHI, E. S., T. L. HICKEY, AND C. W. OYSTER. 1977. Retinogeniculate projections in the rabbit: an autoradiographic study. J. Comp. Neural. 175: I- 12. 34. TAKEDA, T., AND K. MAEKAWA. 1976. The origin of the pretecto-olivary tract. A study using the horseradish peroxidase method. Brain Res. 117: 319-325. 35. TARLOV, E. C., AND R. Y. MOORE. 1966. The tectothalamic connections in the brain of the rabbit. J. Camp. Neurol. 126: 403-422. 36. TERASAWA, K., K. OTANI, AND J. YAMADA. 1979. Descending pathways of the nucleus of the optic tract in the rat. Brain Res. 173: 405-417. 37. WALBERG, F., T. NORDBY, K. P. HOFFMANN, AND H. HOLL~NDER. 1981. Olivary afferents from the pretectal nuclei in the cat. Anat. Embryol. 161: 291-304.