A Cerebello-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 KAZUO ITOH, NOBORU MIZUNO, TETSUO SUGIMOTO, SAKASHI NOMURA, YASUHISA NAKAMURA AND AKIRA KONISHI Department of Anatomy, Faculty of M e d w n e , Kyoto Unruersity, Kyoto fiOfi, Japan
ABSTRACT
A cerebello-pulvino-cortical and a retino-pulvino-cortical pathways were revealed in the cat by means of the horseradish peroxidase (HRP) method. The sites of termination of the cerebellofugal and retinofugal fibers in the pulvinar nucleus (Pul) were visualized by the use of the anterograde transport of HRP. The cerebello-pulvinar fibers were found to arise mainly from the parvicellular region of the lateral cerebellar nucleus and to terminate contralaterally in a narrow area at the extreme dorsolateral edge of the Pul at the level of the stereotaxic frontal plane A-7.0. The area of terminal ramification of the retino-pulvinar fibers was seen as a thin sheet lying at the extreme lateral edge of the Pul through most of the rostrocaudal extent of the Pul, bilaterally with contralateral predominance. The cerebellorecipient area in the Pul did not seem to overlap with the retinorecipient Pul area; the former appeared t o be contiguous ventrolaterally t o the latter. The cerebellorecipient and retinorecipient Pul areas were also observed t o be connected reciprocally with the cerebral cortical areas; the former was connected with the most posterior part of the area 20. and the latter with the area 19.
Trachtenberg, '78) was made into the Pul (7 cats), cerebellar nuclei (11 cats) or cerebral cortex (24 cats) with a 27-gauge needle a t tached t o a 1-pl Hamilton syringe; when the HRP injections into the cerebellar nuclei were performed, the carrier of the microsyringe was inclined caudally 45" in the sagittal plane in order t o avoid penetrating the cerebral hemisphere with the injection-needle. In four cats 0.1 ml of 510% HRP solution was injected unilaterally through the sclera into t h e vitreous body using a 1-ml injection syringe. The cats were sacrificed after 20 t o 48 hours under deep general anesthesia by perfusion MATERIALS AND METHODS through the ascending aorta with 2 1of 7%forThe experiments were performed on 46 malin in 0.1 M phosphate buffer (pH 7.3) (cf. adult cats. Under anesthesia with intraperi- Itoh and Mizuno, '771, followed by 0.5 1 of the toneal sodium pentobarbital (35-40 mg/kgI same buffer containing 10% sucrose. The the cats were placed in a stereotaxic head brains were removed immediately, stored in holder, and a single injection of less than 0.1 the same buffer containing 30%sucrose a t 4°C pl of 50% HRP (Toyobo Grade-I-C, RZ: 3.4) for two t o four days, and cut serially a t 60 pm dissolved in sterile 0.9%saline containing 2% thickness in the frontal plane on a freezing dimethylsulfoxide (Keefer et al., '76) or 0.5% microtome. For the histochemical demonstrapoly-L-ornithine (Itaya et al., '78; Hadley and tion of the enzyme the sections were treated
In the course of our study on efferent and afferent connections of the pulvinar nucleus (Pull in the cat, the cerebellar fibers were found to terminate in the dorsolateral marginal area of the Pul by the horseradish peroxidase (HRPI method. The extreme lateral edge of the Pul of the cat has also been reported to receive the retinal fibers (Berman and Jones, '77).The present communication will describe the direct cerebello-pulvinar projection, and also report the cerebral cortical sites of termination of fibers arising from the cerebellorecipient and the retinorecipient Pul areas.
J. COMP. NEUR.(1979)187: 349-358.
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ITOH. MlZUNO, SUGIMOTO. NOMURA, NAKAMURA A N D KONISHI Ahbreuiattons A , Lamina A of lateral geniculate nucleus A l , Lamina A1 of lateral geniculate nucleus C. Lamina C of lateral geniculate nucleus (21.3. Laminae C 1 X 3 of lateral geniculate nucleus FN. Medial cerebellar nucleus IN. Interpositus cerebellar nucleus L, Lateral sulcus LGN, Dorsal lateral geniculate nucleus LN. Lateral cerebellar nucleus LP. Lateroposterior nucleus of thalamus
MG, Medial geniculate nucleus MIN, Medial interlammar nucleus of lateral geniculate nucleus OT, Optic tract PN, Posterior nucleus of thalamus Pul. Pulvinar nucleus Rh, Rhinal sulcus Sg, Suprageniculate nucleus Sp, Splenial sulcus Say, Suprasylvian S U ~ C U S V . Fourth ventricle
Fig 1 Projection drawings made from a series of frontal sections through the thalamus ( A ) and cerebel lum ( B ) of a representative cat, showing the site of HRP injection (blackened areas in A ) in the lateral part of the Pul and distribution of HRP.labeled neurons (black dots in B) in the lateral cerebellar nucleus (LNI contralateral to t h e injection
with 3,3'-diaminobenzidine tetrahydrochloride as described by Streit and Reubi ('77) or with benzidine through a procedure modified slightly from t h a t described by De Olmos ('77). After the enzyme reaction was completed, t h e sections were mounted onto gelatinized slides, counterstained with 1X) cresylecht violet for diaminobenzidine reaction or with 1% neutral red for benzidine reaction, and examined microscopically under darkfield and bright-field illumination. RESULTS
In three of seven cats injected with HRP into
t h e Pul, HRP-labeled neurons were found in the lateral cerebellar nucleus (LCN! contralateral to t h e injected site. The H R P injection in these cats was centered on t h e lateral part of t h e Pul a t the level of t h e stereotaxic frontal plane A-7.0, and t h e spread of injected HRP involved more or less the dorsolateral marginal area of t h e Pul (fig. 1 A ) . HRP-labeled nerve cells seen in t h e LCN of these cats were not numerous and were localized in t h e ventral region of t h e LCN at t h e middle levels of t h e rostrocaudal extent of t h e LCN (figs. lB, 2). In 11cats t h e H R P injection was attempted
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Fig. 2 A dark-field photomicrograph of neurons in the parvicellular region of t h e lateral cerebellar n u cleus, which are labeled with HRP injected contralaterally into the dorsolateral marginal area of the Pul. The level of the lateral cerebellar nucleus corresponds to t h e drawing 3 in the figure 1B. x 125.
Fig. 3 A. Projection drawings of frontal sections through the HRP injection site (areas marked with crosses) centered on the parvicellular region of t h e lateral cerebellar nucleus of a representative cat, in which t h e cerebello-pulvinar fiber terminals were labeled anterogradely with HRP in the dorsal marginal area of the Pul contralateral to t h e injection. B. Diagrammatic representation of distribution pattern of the cerebellar (crosses) and retinal (dots) fiber terminals in t h e Pul and LGN.
unilaterally into the cerebellar nuclei. In four of them, in which the HRP injection was cen-
tered on the parvicellular region of the LCN (fig. 3A), possible axon terminals labeled with HRP transported anterogradely (cf. Lynch et al., '73, '74; Scalia and Colman, '74; Reperant,
' 7 5 ; Colman et al., '76; Mesulam, '76, '78; De Olmos, '77; Hardy and Heimer, '77; Robertson, '77; Streit and Reubi, '77) were seen contralaterally in the dorsolateral marginal area of the Pul. The area of distribution of these axon terminals was observed to take the form of a
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Fig. 4 Dark-field photomicrographs of HRP-labeled cerebello-pulvinar (A) and retino-pulvinar (B) fiber terminals. Compare with the figure 3B. In B. HRP-labeled retino-geniculate fiber terminals are also seen a t the right lower corner of the figure. A x 75, B x 40
narrow band along the dorsolateral border of the Pul and were clearly separated from the reticular nucleus of the thalamus with the external medullary lamina of the thalamus; it could be seen a t most in four of serial sections of 60 p m thickness a t the level of the stereotaxic frontal plane A-7.0 (figs. 3B, 4A). This cerebellorecipient area in the Pul appeared to be located closely to the retinorecipient Pul area reported by Berman and Jones ('77). In order to examine whether the cerebellorecipient Pul area overlapped with the retinorecipient Pul area, the following experiments were carried out: In four cats injected with HRP unilaterally into the vitreous body, possible terminals of retinofugal fibers were labeled anterogradely with the enzyme. They were seen bilaterally with a contralateral predominqnce in the Pul as well as in the dorsal and ventral lateral geniculate nuclei, superior colliculus, pretectum and suprachiasmatic nucleus, and also contralaterally in the nuclei of the accessory optic tract. This pattern of distribution of retinofugal fibers was identical with that reported in the previous studies by other meth-
ods (cf. Meikle and Sprague. '64; Singleton and Peele, '65; Garey and Powell, '68; Guillery, '70; Hendrickson et al., '72; Moore, '73; Hickey and Guillery, '74; Graybiel, '75; Hollander and Sanides, '76; Berman, '77). In good agreement with the description of Berman and Jones ('77) who first used the autoradiographic tracing method to demonstrate a retino-pulvinar projection in the cat, the area of distribution of HRP-labeled terminals of the retino-pulvinar fibers was seen as a thin sheet lying a t the extreme lateral edge of the Pul, separated clearly from the reticular nucleus of the thalamus with the external medullary lamina of t h e thalamus, and extending through most of the rostrocaudal extent of the Pul (figs. 3B, 4B). The distribution area of the retino-pulvinar fibers, however, did not seem to overlap with that of the cerebello-pulvinar fibers; the latter appeared to be contiguous ventrolaterally to the former (fig. 3B). In the next step, the cerebral cortical sites of termination of projection fibers arising from the cerebellorecipient and the retinorecipient Pul areas were examined by the retrograde HRP method. In 24 cats injected with
CEREBELLO-PULVINO-CORTICAL PATHWAY IN THE CAT
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C
Fig. 5 Distribution pattern of neurons labeled with HRP injected into the area 20 and area 19. A. Posterior view of the cerebral hemisphere to show the projection area (dotted area) of fibers arising from the cerebellorecipient Pul area. B. Projection drawing of a frontal section through the HRP injection site (blackened area) in t h e area 20 of a representative cat, in which neurons in the cerebellorecipient Pul area were labeled retrogradely with HHP. C. HRP injection sites in the area 19 are indicated by triangles. Neurons in the retinorecipient Pul area were labeled retrogradely with HRP injected in these cortical areas. D. Diagrammatic representation of distribution pattern of neurons labeled retrogradely with HRP injected into the area 20 and area 19. Black dots represent the area of distribution of neurons labeled with HRP injected into t h e cortical area shown in A and B. Black triangles represent the distribution area of neurons labeled with HRP injected into t h e cortical area pointed by a black triangle in C. Compare with the figure 3B.
HRP into various areas in t h e cerebral cortex, t h e pattern of distribution of HRP-labeled nerve cells within the Pul was studied. The nerve cells within t h e cerebellorecipient area of t h e Pul were labeled with H R P when t h e HRP injection was centered on t h e posterior
portions of t h e hippocampal fusiform gyrus of Papez ('29) (4 cats) (figs. SA,B,D); HRP-labeled neurons were surrounded with possible axon terminals labeled anterogradely with HRP (fig. 6A). On t h e other hand, the Pul neurons within t h e retinorecipient Pul area were
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Fig. 6 Dark-field photomicrographs of nerve cells in the cerebellorecipient (A) and retinorecipient (B) Pul areas; these nerve cells are labeled retrogradely with HRP injected into the area 20 or area 19, respectively. Axon terminals labeled anterogradely with HRP are also seen arnund the HRP-labeled nerve cells. X 40.
labeled retrogradely with HRP injected into cerebral cortical areas around the lateral or the posterior lateral sulcus (8 cats) (figs. 5C,D) ; possible axon terminals labeled anterogradely with HRP were also seen around the HRP-labeled nerve cells (fig. 6B). In conclusion, the cerebello-pulvinar path differs from the retino-pulvinar path both in its cortical target and in its relay zones in the Pul. DISCUSSION
The present study showed that some neurons in the ventral part of the LCN sent their axons contralaterally to the Pul. These LCN neurons were mainly located within and around the subnucleus lateralis parvicellularis of Flood and Jansen ('61). The ventral region of the LCN has been reported to receive input mainly from the flocculonodular lobe (Voogd, '64; Angaut and Brodal, '67; Haines, '77b; Yamamoto, '78) as well as primary and secondary vestibulocerebellar fibers (Brodal and Hdivik, '64; Carpenter et al., '721, and to project directly to the oculomotor and trochlear nuclei through the superior cerebellar
peduncle (Carpenter and Strominger, '64; Chan-Palay, '77). The ventral region of the LCN may also receive input of visual origin via the pretecto-olivo (dorsal cap)-cerebellar pathway (cf. Mizuno et al., '73, '74; Mizuno and Itoh, '75; Maekawa and Takeda, '76; Takeda and Maekawa, '76; Itoh, '77). The evidence for this pathway comes from our observations of HRP-labeled cells in the dorsal cap of the inferior olive after HRP was injected contralaterally into the ventral region of the LCN (unpublished data). Haines ('77a) considered that t h e available data implicated the ventral region of the LCN as relay centers for cerebellar and vestibular modulation of eyemovements. The recent HRP studies in the cat and monkey did not support this notion; no HRP-labeled nerve cells were described in the LCN after injection of HRP into the oculomotor nucleus (Graybiel and Hartwieg, '74; Gacek, '77; Steiger and Buttner-Ennever,'79). The possibility of termination of the cerebellar fibers within the oculomotor nucleus, however, did not seem to be excluded completely (cf. Carpenter and Strominger, '64; ChanPalay, '77).
CEREBEL1.0 PIJLVINO-CORTICAL PATHWAY IN THE CAT
The Pul of t h e cat has been reported to receive afferent fibers from the visual cortex (Heath and Jones, '71; Kawamura et al., '74; Updyke, '77), pretectum (Graybiel, '72; Berman, '77; Itoh, '77) and retina (Berman and Jones, '77). The terminal areas of these afferent fibers to t h e Pul have been shown to form a n orderly sequence of adjoining, well-defined subfields in t h e Pul (Berson and Graybiel, '78). The cerebello-pulvinar fibers found in t h e present study terminated contralaterally in a localized, narrow area along t h e dorsolateral margin of t h e Pul at t h e level of t h e stereotaxic frontal plane A-7.0. This area lying at t h e extreme dorsolateral edge of t h e Pul appeared to be contiguous ventrolaterally to the area of terminal ramification of the retino-pulvinar fibers. The data presented by Updyke ('77) and Berson and Graybiel ('78) have suggested t h a t t h e retinorecipient area of the Pul receives afferent fibers from t h e area 19 of t h e cerebral cortex in t h e cat. The present study indicated t h a t t h e retinorecipient Pul area was connected reciprocally with the cerebral cortical areas around t h e lateral and t h e posterior lateral sulci; these areas appeared to be included in the area 19 of Otsuka and Hassler ('62). The area of termination of the cerebellopulvinar fibers did not seem to overlap with the Pul area receiving afferent fibers from t h e retina, prelectum and visual cortex (cf. Berman and Jones, '77; Berson and Graybiel, '78). In a study by the autoradiographic tracer method, Updyke ('77) showed t h a t a posterior portion of t h e area 20 in the cat sent fibers to t h e dorsolateral marginal area of t h e Pul; t h e locus and extent of this Pul area were similar to those of t h e site of termination of t h e cerebello-pulvinar fibers found in t h e present study (cf. case 35 L shown in fig. 15of Updyke, '77?. The present study suggested t h a t t h e cerebellorecipient Pul area was connected reciprocally with the posterior part of t h e hippocampal fusiform gyrus of Papez ('291; this cortical area appeared to be located within the posterior part of t h e paralimbic region of Sanides and Hoffmann ('69) or t h e area 20 of Heath and Jones ('71). The available data, thus, implicate t h a t a certain amount of t h e data relayed to t h e area 20 via t h e cerebello-pulvino-corticalpathway may have its origin from vestibular and/or retinal input. The area 20 in t h e monkey appears to play important roles in the visual dis-
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crimination (Iwai and Mishkin, '69; Cowey and Gross, '70; Gross et al.. '71; Blake et al., '77') or identification (Wilson e t al., '72), and has been known to receive afferent fibers from t h e Pul (Benevento and Rezak, '76; Trojanowski and ,Jacobson, '76). The functional meaning of t h e cerebello-pulvino-cortical pathway found in t h e cat, however, still remains t o be elucidated. ACKNOWLEDGMENTS
The photographic work of Mr. Akira Uesugi is gratefully acknowledged. LITERATURE CITED Angaut, P., and A. Brodal 1967 The projection of the 'vestibulocerebellum' onto t h e vestibular nuclei in the cat. Arch. ital. Biol., 105: 441-479. Renevcnto, L. A,. and M. Rezak 1976 The cortical projections of' the inferior pulvinar and adjacent lateral pulvinar in the rhesus monkey (Mncara mulatta): a n autoradiographic study. Brain Res., 108: 1-24. Berman, N. 1977 Connections of the pretectum in the cat. J. Comp. Neur., 174: 227-254. Berman, N., and E. G. Jones 1977 A retino-pulvinar projection in the cat. Brain Res., 134: 237-248. Berson, D. M., and A. hl. Graybiel 1978 Parallel thalamic zones in the LP-pulvinar complex of the cat identified by their afferent and efferent connections. Brain Res., 147: 139-146. Blake, L., C. D. Jarvis and M. Mishkin 1977 Pattern discrimination thresholds after partial inferior temporal or lateral striate lesions in monkeys. Brain Res., 120: 209-220. Brodal, A., and B. Heivik 1964 Site and mode of termination of primary vestibulocerebellar fibers in the cat. Arch. ital. Biol., 102: 1-21. Carpenter, M. B., B. M. Stein and P. Peter 1972 Primary vestibulocerebellar fibers in the monkey. Distribution of fibers arising from distinctive cell groups of the vestibular ganglia. Am. J. Anat., 135: 221-250. Carpenter, LM.B., and N. L. Strominger 1964 Cerebellooculomotor fibers in the rhesus monkey. J. Comp. Neur., 123: 211-230. Chan-Palay, V. 1977 Cerebellar Dentate Nucleus. Organization, Cytology and Transmitters. Springer, Berlin, p. 548. Colman, D. R., F. Scalia and E. Cabrales 1976 Light and electron microscopic observations on the anterograde transport of horseradish peroxidase in the optic pathways in the mouse and rat. Brain Res., 102: 156.163. Cowey, A., and C. G. Gross 1970 Effects of foveal prestriate and inferotemporal lesions on visual discrimination by rhesus monkeys. Exp. Brain Res., 11: 128-144. De Olmos. J . S. 1977 An improved HRP method for t h e study of central nervous connections. Exp. Brain Res., 29: 541-551. Flood, S., and J. Jansen 1961 On the cerebellar nuclei in the cat. Acta anat., 46: 52-72. Gacek, R. R. 1977 Location of brain stem neurons projecting to t h e oculomotor nucleus in the cat. Exp. Neurol., 57: 725-749. Garey, L. J., and T. P. S. Powell 1968 The projection of the retina in the cat. J. Anat. (London), 102: 189-222.
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Graybiel. A. M. 1972 Some extrageniculate visual pathways in the cat. Invest. Ophthal., 11: 322-332. 1975 Anatomical organization of retinotectal afferents in the cat: an autoradiographic study. Brain Res., 96: 1-23. Graybiel, A. M., and E. A. Hartwieg 1974 Some afferent connections of the oculomotor complex in t he cat: an experimental study with tracer techniques. Brain Res., 81: 543-551. Gross, C. G., A. Cowey and F. J. Manning 1971 Further analysis of visual discrimination deficits following foveal prestriate and inferotemporal lesions in rhesus monkeys. J. Comp. Physiol. Psychol., 76: 1-7. Guillery, R. W. 1970 The laminar distribution of retinal fibers in t h e dorsal lateral geniculate nucleus of the cat: a new interpretation. J. Comp. Neur., 138: 339-368. Hadley, R. T., and M. C. Trachtenberg 1978 Poly-Lornithine enhances the uptake of horseradish peroxidase. Brain Res., 158: 1-14. Haines, D. E. 1977a A proposed functional significance of parvicellular regions of the lateral and medial cerebellar nuclei. Brain Behav. and Evol., 14: 328-340. 1977b Cerebellar corticonuclear and corticovestibular fibers of th e flocculonodular lobe in a prosimian primate (Galago senegalensis). J. Comp. Neur.. 174: 607-630. Hardy, H., and L. Heimer 1977 A safer and more sensitive substitute for diaminobenzidine in the light microscopic demonstration of retrograde and anterograde axonal transport of HRP. Neurosci. Lett., 5: 235-240. Heath, C. J . , and E. G. Jones 1971 The anatomical organization of the suprasylvian gyrus of the cat. Ergebn. Anat. Entwicklungsgesch., 45: 1-64. Hendrickson, A. E., N. Wagoner and W. M. Cowan 1972 An autoradiographic and electron microscopic study of retino-hypothalamic connections. 2. Zellforsch., 135: 1-26. Hickey, T. L., and R. W. Guillery 1974 An autoradiographic study of retinogeniculate pathways in t he cat and the fox. J. Comp. Neur., 156: 239-254. Hollander, H., and D. Sanides 1976 The retinal projection to the ventral part of t h e lateral geniculate nucleus. An experimental study with silver-impregnation methods and axoplasmic protein tracing. Exp. Brain Res., 26: 329-342. Itaya, S. K., T . H. Williams and E. L. Engel 1978 Anterograde transport of horseradish peroxidase enhanced by poly-L-ornithine. Brain Res., 150: 170-176. Itoh. K. 1977 Efferent projections of the pretectum in th e cat. Exp. Brain Res., 30: 89-105. Itoh, K., and N. Mizuno 1977 Topographical arrangement of thalamocortical neurons in the centrolateral nucleus E L ) of t h e cat, with special reference t o a spino-thalamomotor cortical path through the CL. Exp. Brain Res., 30: 471-480. Iwai, E., and M. Mishkin 1969 Further evidence on t he locus of th e visual area in the temporal lobe of the monkey. Exp. Neurol., 25: 585-594. Kawamura, S. 1974 Topical organization of t he extrageniculate visual system in t he cat. Exp. Neurol., 45: 451-461. Kawamura, S., J. M. Sprague and K. Niimi 1974 Corticofugal projections from the visual cortices to the thalamus. pretectum and superior colliculus in the cat. J. Comp. Neur., 158: 339-362. Keefer, D. A., W. B. Spatz and U. Misgeld 1976 Golgi-like staining of neocortical neurons using retrogradely transport horseradish peroxidase. Neurosci. Lett., 3: 233-237. Lynch, G., C. Gall, P. Mensah and C. W. Cotman 1974 Horseradish peroxidase histochemistry: anew method for
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tracing efferent projections in the central nervous system. Brain Res., 65: 373-380. Lynch, G., R. L. Smith, P. Mensah and C. Cotman 1973 Tracing the dentate gyrus mossy fiber system with horseradish peroxidase histochemistry. Exp. Neurol., 40: 516-524. Maekawa, K., and T. Takeda 1976 Electrophysiological identification of the climbing and mossy fiber pathways from the rabbit's retina to the contralateral cerebellar flocculus. Brain Res., 109: 169-174. Meikle, T . H., and J. M. Sprague 1964 The neural organization of the visual pathways in the cat. Int. Rev. Neurobiol., 6: 149-189. Mesulam, M.-M. 1976 The blue reaction product in horseradish peroxidase neurohistochemistry : incubation parameters and visibility. J. Histochem. Cytochem., 24: 1273-1280. 1978 Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neuronal afferents and efferents. J. Histochem. Cytochem., 26: 106117. Mizuno, N., andK. Itoh 1975 Light and electron microscopical studies of pretecto-olivary projections in the rabbit and cat. J. physiol. SOC. Jap., 37: 374-375. Mizuno, N., K. Mochizuki, C. Akimoto and R. Matsushima 1973 Pretectal projections to the inferior alive 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 pretecto-olivary fibers. Brain Res., 77: 385-395. Moore, R. Y. 1973 Retinohypothalamic projection in mammals: a comparative study. Brain Res., 49: 403-409. Otsuka, R., and R. Hassler 1962 Uber Auf'bau und Gliederung der corticalen SehsphaTe bei der Katze. Arch. Psychiat. Nervenkrankh., 203: 212-234. Papez, J. W. 1929 Comparative Neurology. A Manual and Text far the Study of the Nervous System of Vertebrates. Crowell, New York, p. 518. R e g r a n t , J. 1975 The orthograde transport of horseradish peroxidase in the visual system. Brain Res., 85: 307-312. Robertson, R. T. 1977 Bidirectional movement of horseradish peroxidase and the demonstration of reciprocal thalamocortical connections. Brain Res., 130: 538-544. Sanides, F., and J. Hoffmann 1969 Cyto- and myeloarchitecture of the visual cortex of the cat and of the surrounding integration cortices. J. Hirnforsch., 11: 79-104. Scalia, F., and D. R. Colman 1974 Aspects of the central projection of the optic nerve in the frog as revealed by anterograde migration of horseradish peroxidase. Brain Res., 79: 496-504. Singleton, M. C., and T. L. Peele 1965 Distribution of optic fibers in the cat. J. Comp. Neur., 125: 303-328. Steiger, H. J.,and J. A. Buttner-Ennever 1979 Oculomotor nucleus afferents in the monkey demonstrated with horseradish peroxidase. Brain Res., 160: 1-15. Streit, P., and J. C. Reubi 1977 A new and sensitive staining method for axonally transported horseradish peroxidase (HRP) in t.he pigeon visual system. Brain Res., 126: 530-537. 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. Trojanowski, J. Q , ,and S. Jacobson 1976 Areal and laminar distribution of some pulvinar cortical efferents in rhesus monkey. J. Comp. Neur., 169: 371-392. Updyke, B. V . 1977 Topographic organization of the pro-
CEREBELLO-PULVINO-CORTICAL PATHWAY IN THE CAT jections from cortical areas 17, 18, and 19 onto the thalamus, pretectum and superior colliculus in the cat. J. Comp. Neur., 173; 81-122. Voogd, J. 1964 The Cerebellum of t h e Cat. Structure and Fibre Connexions. Van Gorcum, Assen, p. 215. Wilson, M., H. M. Kaufman, R. E. Zieler and J. P. Lieb 1972 Visual identifications and memory in monkeys with cir-
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ABSTRACT
A cerebello-pulvino-cortical and a retino-pulvino-cortical pathways were revealed in the cat by means of the horseradish peroxidase (HRP) method. The sites of termination of the cerebellofugal and retinofugal fibers in the pulvinar nucleus (Pul) were visualized by the use of the anterograde transport of HRP. The cerebello-pulvinar fibers were found to arise mainly from the parvicellular region of the lateral cerebellar nucleus and to terminate contralaterally in a narrow area at the extreme dorsolateral edge of the Pul at the level of the stereotaxic frontal plane A-7.0. The area of terminal ramification of the retino-pulvinar fibers was seen as a thin sheet lying at the extreme lateral edge of the Pul through most of the rostrocaudal extent of the Pul, bilaterally with contralateral predominance. The cerebellorecipient area in the Pul did not seem to overlap with the retinorecipient Pul area; the former appeared t o be contiguous ventrolaterally t o the latter. The cerebellorecipient and retinorecipient Pul areas were also observed t o be connected reciprocally with the cerebral cortical areas; the former was connected with the most posterior part of the area 20. and the latter with the area 19.
Trachtenberg, '78) was made into the Pul (7 cats), cerebellar nuclei (11 cats) or cerebral cortex (24 cats) with a 27-gauge needle a t tached t o a 1-pl Hamilton syringe; when the HRP injections into the cerebellar nuclei were performed, the carrier of the microsyringe was inclined caudally 45" in the sagittal plane in order t o avoid penetrating the cerebral hemisphere with the injection-needle. In four cats 0.1 ml of 510% HRP solution was injected unilaterally through the sclera into t h e vitreous body using a 1-ml injection syringe. The cats were sacrificed after 20 t o 48 hours under deep general anesthesia by perfusion MATERIALS AND METHODS through the ascending aorta with 2 1of 7%forThe experiments were performed on 46 malin in 0.1 M phosphate buffer (pH 7.3) (cf. adult cats. Under anesthesia with intraperi- Itoh and Mizuno, '771, followed by 0.5 1 of the toneal sodium pentobarbital (35-40 mg/kgI same buffer containing 10% sucrose. The the cats were placed in a stereotaxic head brains were removed immediately, stored in holder, and a single injection of less than 0.1 the same buffer containing 30%sucrose a t 4°C pl of 50% HRP (Toyobo Grade-I-C, RZ: 3.4) for two t o four days, and cut serially a t 60 pm dissolved in sterile 0.9%saline containing 2% thickness in the frontal plane on a freezing dimethylsulfoxide (Keefer et al., '76) or 0.5% microtome. For the histochemical demonstrapoly-L-ornithine (Itaya et al., '78; Hadley and tion of the enzyme the sections were treated
In the course of our study on efferent and afferent connections of the pulvinar nucleus (Pull in the cat, the cerebellar fibers were found to terminate in the dorsolateral marginal area of the Pul by the horseradish peroxidase (HRPI method. The extreme lateral edge of the Pul of the cat has also been reported to receive the retinal fibers (Berman and Jones, '77).The present communication will describe the direct cerebello-pulvinar projection, and also report the cerebral cortical sites of termination of fibers arising from the cerebellorecipient and the retinorecipient Pul areas.
J. COMP. NEUR.(1979)187: 349-358.
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ITOH. MlZUNO, SUGIMOTO. NOMURA, NAKAMURA A N D KONISHI Ahbreuiattons A , Lamina A of lateral geniculate nucleus A l , Lamina A1 of lateral geniculate nucleus C. Lamina C of lateral geniculate nucleus (21.3. Laminae C 1 X 3 of lateral geniculate nucleus FN. Medial cerebellar nucleus IN. Interpositus cerebellar nucleus L, Lateral sulcus LGN, Dorsal lateral geniculate nucleus LN. Lateral cerebellar nucleus LP. Lateroposterior nucleus of thalamus
MG, Medial geniculate nucleus MIN, Medial interlammar nucleus of lateral geniculate nucleus OT, Optic tract PN, Posterior nucleus of thalamus Pul. Pulvinar nucleus Rh, Rhinal sulcus Sg, Suprageniculate nucleus Sp, Splenial sulcus Say, Suprasylvian S U ~ C U S V . Fourth ventricle
Fig 1 Projection drawings made from a series of frontal sections through the thalamus ( A ) and cerebel lum ( B ) of a representative cat, showing the site of HRP injection (blackened areas in A ) in the lateral part of the Pul and distribution of HRP.labeled neurons (black dots in B) in the lateral cerebellar nucleus (LNI contralateral to t h e injection
with 3,3'-diaminobenzidine tetrahydrochloride as described by Streit and Reubi ('77) or with benzidine through a procedure modified slightly from t h a t described by De Olmos ('77). After the enzyme reaction was completed, t h e sections were mounted onto gelatinized slides, counterstained with 1X) cresylecht violet for diaminobenzidine reaction or with 1% neutral red for benzidine reaction, and examined microscopically under darkfield and bright-field illumination. RESULTS
In three of seven cats injected with HRP into
t h e Pul, HRP-labeled neurons were found in the lateral cerebellar nucleus (LCN! contralateral to t h e injected site. The H R P injection in these cats was centered on t h e lateral part of t h e Pul a t the level of t h e stereotaxic frontal plane A-7.0, and t h e spread of injected HRP involved more or less the dorsolateral marginal area of t h e Pul (fig. 1 A ) . HRP-labeled nerve cells seen in t h e LCN of these cats were not numerous and were localized in t h e ventral region of t h e LCN at t h e middle levels of t h e rostrocaudal extent of t h e LCN (figs. lB, 2). In 11cats t h e H R P injection was attempted
CEREBELLO-PULVINO-CORTICAL PATHWAY IN THE CAT
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Fig. 2 A dark-field photomicrograph of neurons in the parvicellular region of t h e lateral cerebellar n u cleus, which are labeled with HRP injected contralaterally into the dorsolateral marginal area of the Pul. The level of the lateral cerebellar nucleus corresponds to t h e drawing 3 in the figure 1B. x 125.
Fig. 3 A. Projection drawings of frontal sections through the HRP injection site (areas marked with crosses) centered on the parvicellular region of t h e lateral cerebellar nucleus of a representative cat, in which t h e cerebello-pulvinar fiber terminals were labeled anterogradely with HRP in the dorsal marginal area of the Pul contralateral to t h e injection. B. Diagrammatic representation of distribution pattern of the cerebellar (crosses) and retinal (dots) fiber terminals in t h e Pul and LGN.
unilaterally into the cerebellar nuclei. In four of them, in which the HRP injection was cen-
tered on the parvicellular region of the LCN (fig. 3A), possible axon terminals labeled with HRP transported anterogradely (cf. Lynch et al., '73, '74; Scalia and Colman, '74; Reperant,
' 7 5 ; Colman et al., '76; Mesulam, '76, '78; De Olmos, '77; Hardy and Heimer, '77; Robertson, '77; Streit and Reubi, '77) were seen contralaterally in the dorsolateral marginal area of the Pul. The area of distribution of these axon terminals was observed to take the form of a
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Fig. 4 Dark-field photomicrographs of HRP-labeled cerebello-pulvinar (A) and retino-pulvinar (B) fiber terminals. Compare with the figure 3B. In B. HRP-labeled retino-geniculate fiber terminals are also seen a t the right lower corner of the figure. A x 75, B x 40
narrow band along the dorsolateral border of the Pul and were clearly separated from the reticular nucleus of the thalamus with the external medullary lamina of the thalamus; it could be seen a t most in four of serial sections of 60 p m thickness a t the level of the stereotaxic frontal plane A-7.0 (figs. 3B, 4A). This cerebellorecipient area in the Pul appeared to be located closely to the retinorecipient Pul area reported by Berman and Jones ('77). In order to examine whether the cerebellorecipient Pul area overlapped with the retinorecipient Pul area, the following experiments were carried out: In four cats injected with HRP unilaterally into the vitreous body, possible terminals of retinofugal fibers were labeled anterogradely with the enzyme. They were seen bilaterally with a contralateral predominqnce in the Pul as well as in the dorsal and ventral lateral geniculate nuclei, superior colliculus, pretectum and suprachiasmatic nucleus, and also contralaterally in the nuclei of the accessory optic tract. This pattern of distribution of retinofugal fibers was identical with that reported in the previous studies by other meth-
ods (cf. Meikle and Sprague. '64; Singleton and Peele, '65; Garey and Powell, '68; Guillery, '70; Hendrickson et al., '72; Moore, '73; Hickey and Guillery, '74; Graybiel, '75; Hollander and Sanides, '76; Berman, '77). In good agreement with the description of Berman and Jones ('77) who first used the autoradiographic tracing method to demonstrate a retino-pulvinar projection in the cat, the area of distribution of HRP-labeled terminals of the retino-pulvinar fibers was seen as a thin sheet lying a t the extreme lateral edge of the Pul, separated clearly from the reticular nucleus of the thalamus with the external medullary lamina of t h e thalamus, and extending through most of the rostrocaudal extent of the Pul (figs. 3B, 4B). The distribution area of the retino-pulvinar fibers, however, did not seem to overlap with that of the cerebello-pulvinar fibers; the latter appeared to be contiguous ventrolaterally to the former (fig. 3B). In the next step, the cerebral cortical sites of termination of projection fibers arising from the cerebellorecipient and the retinorecipient Pul areas were examined by the retrograde HRP method. In 24 cats injected with
CEREBELLO-PULVINO-CORTICAL PATHWAY IN THE CAT
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C
Fig. 5 Distribution pattern of neurons labeled with HRP injected into the area 20 and area 19. A. Posterior view of the cerebral hemisphere to show the projection area (dotted area) of fibers arising from the cerebellorecipient Pul area. B. Projection drawing of a frontal section through the HRP injection site (blackened area) in t h e area 20 of a representative cat, in which neurons in the cerebellorecipient Pul area were labeled retrogradely with HHP. C. HRP injection sites in the area 19 are indicated by triangles. Neurons in the retinorecipient Pul area were labeled retrogradely with HRP injected in these cortical areas. D. Diagrammatic representation of distribution pattern of neurons labeled retrogradely with HRP injected into the area 20 and area 19. Black dots represent the area of distribution of neurons labeled with HRP injected into t h e cortical area shown in A and B. Black triangles represent the distribution area of neurons labeled with HRP injected into t h e cortical area pointed by a black triangle in C. Compare with the figure 3B.
HRP into various areas in t h e cerebral cortex, t h e pattern of distribution of HRP-labeled nerve cells within the Pul was studied. The nerve cells within t h e cerebellorecipient area of t h e Pul were labeled with H R P when t h e HRP injection was centered on t h e posterior
portions of t h e hippocampal fusiform gyrus of Papez ('29) (4 cats) (figs. SA,B,D); HRP-labeled neurons were surrounded with possible axon terminals labeled anterogradely with HRP (fig. 6A). On t h e other hand, the Pul neurons within t h e retinorecipient Pul area were
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ITOH, MIZUNO, SUGIMOTO, NOMURA, NAKAMURA AND KONISHI
Fig. 6 Dark-field photomicrographs of nerve cells in the cerebellorecipient (A) and retinorecipient (B) Pul areas; these nerve cells are labeled retrogradely with HRP injected into the area 20 or area 19, respectively. Axon terminals labeled anterogradely with HRP are also seen arnund the HRP-labeled nerve cells. X 40.
labeled retrogradely with HRP injected into cerebral cortical areas around the lateral or the posterior lateral sulcus (8 cats) (figs. 5C,D) ; possible axon terminals labeled anterogradely with HRP were also seen around the HRP-labeled nerve cells (fig. 6B). In conclusion, the cerebello-pulvinar path differs from the retino-pulvinar path both in its cortical target and in its relay zones in the Pul. DISCUSSION
The present study showed that some neurons in the ventral part of the LCN sent their axons contralaterally to the Pul. These LCN neurons were mainly located within and around the subnucleus lateralis parvicellularis of Flood and Jansen ('61). The ventral region of the LCN has been reported to receive input mainly from the flocculonodular lobe (Voogd, '64; Angaut and Brodal, '67; Haines, '77b; Yamamoto, '78) as well as primary and secondary vestibulocerebellar fibers (Brodal and Hdivik, '64; Carpenter et al., '721, and to project directly to the oculomotor and trochlear nuclei through the superior cerebellar
peduncle (Carpenter and Strominger, '64; Chan-Palay, '77). The ventral region of the LCN may also receive input of visual origin via the pretecto-olivo (dorsal cap)-cerebellar pathway (cf. Mizuno et al., '73, '74; Mizuno and Itoh, '75; Maekawa and Takeda, '76; Takeda and Maekawa, '76; Itoh, '77). The evidence for this pathway comes from our observations of HRP-labeled cells in the dorsal cap of the inferior olive after HRP was injected contralaterally into the ventral region of the LCN (unpublished data). Haines ('77a) considered that t h e available data implicated the ventral region of the LCN as relay centers for cerebellar and vestibular modulation of eyemovements. The recent HRP studies in the cat and monkey did not support this notion; no HRP-labeled nerve cells were described in the LCN after injection of HRP into the oculomotor nucleus (Graybiel and Hartwieg, '74; Gacek, '77; Steiger and Buttner-Ennever,'79). The possibility of termination of the cerebellar fibers within the oculomotor nucleus, however, did not seem to be excluded completely (cf. Carpenter and Strominger, '64; ChanPalay, '77).
CEREBEL1.0 PIJLVINO-CORTICAL PATHWAY IN THE CAT
The Pul of t h e cat has been reported to receive afferent fibers from the visual cortex (Heath and Jones, '71; Kawamura et al., '74; Updyke, '77), pretectum (Graybiel, '72; Berman, '77; Itoh, '77) and retina (Berman and Jones, '77). The terminal areas of these afferent fibers to t h e Pul have been shown to form a n orderly sequence of adjoining, well-defined subfields in t h e Pul (Berson and Graybiel, '78). The cerebello-pulvinar fibers found in t h e present study terminated contralaterally in a localized, narrow area along t h e dorsolateral margin of t h e Pul at t h e level of t h e stereotaxic frontal plane A-7.0. This area lying at t h e extreme dorsolateral edge of t h e Pul appeared to be contiguous ventrolaterally to the area of terminal ramification of the retino-pulvinar fibers. The data presented by Updyke ('77) and Berson and Graybiel ('78) have suggested t h a t t h e retinorecipient area of the Pul receives afferent fibers from t h e area 19 of t h e cerebral cortex in t h e cat. The present study indicated t h a t t h e retinorecipient Pul area was connected reciprocally with the cerebral cortical areas around t h e lateral and t h e posterior lateral sulci; these areas appeared to be included in the area 19 of Otsuka and Hassler ('62). The area of termination of the cerebellopulvinar fibers did not seem to overlap with the Pul area receiving afferent fibers from t h e retina, prelectum and visual cortex (cf. Berman and Jones, '77; Berson and Graybiel, '78). In a study by the autoradiographic tracer method, Updyke ('77) showed t h a t a posterior portion of t h e area 20 in the cat sent fibers to t h e dorsolateral marginal area of t h e Pul; t h e locus and extent of this Pul area were similar to those of t h e site of termination of t h e cerebello-pulvinar fibers found in t h e present study (cf. case 35 L shown in fig. 15of Updyke, '77?. The present study suggested t h a t t h e cerebellorecipient Pul area was connected reciprocally with the posterior part of t h e hippocampal fusiform gyrus of Papez ('291; this cortical area appeared to be located within the posterior part of t h e paralimbic region of Sanides and Hoffmann ('69) or t h e area 20 of Heath and Jones ('71). The available data, thus, implicate t h a t a certain amount of t h e data relayed to t h e area 20 via t h e cerebello-pulvino-corticalpathway may have its origin from vestibular and/or retinal input. The area 20 in t h e monkey appears to play important roles in the visual dis-
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crimination (Iwai and Mishkin, '69; Cowey and Gross, '70; Gross et al.. '71; Blake et al., '77') or identification (Wilson e t al., '72), and has been known to receive afferent fibers from t h e Pul (Benevento and Rezak, '76; Trojanowski and ,Jacobson, '76). The functional meaning of t h e cerebello-pulvino-cortical pathway found in t h e cat, however, still remains t o be elucidated. ACKNOWLEDGMENTS
The photographic work of Mr. Akira Uesugi is gratefully acknowledged. LITERATURE CITED Angaut, P., and A. Brodal 1967 The projection of the 'vestibulocerebellum' onto t h e vestibular nuclei in the cat. Arch. ital. Biol., 105: 441-479. Renevcnto, L. A,. and M. Rezak 1976 The cortical projections of' the inferior pulvinar and adjacent lateral pulvinar in the rhesus monkey (Mncara mulatta): a n autoradiographic study. Brain Res., 108: 1-24. Berman, N. 1977 Connections of the pretectum in the cat. J. Comp. Neur., 174: 227-254. Berman, N., and E. G. Jones 1977 A retino-pulvinar projection in the cat. Brain Res., 134: 237-248. Berson, D. M., and A. hl. Graybiel 1978 Parallel thalamic zones in the LP-pulvinar complex of the cat identified by their afferent and efferent connections. Brain Res., 147: 139-146. Blake, L., C. D. Jarvis and M. Mishkin 1977 Pattern discrimination thresholds after partial inferior temporal or lateral striate lesions in monkeys. Brain Res., 120: 209-220. Brodal, A., and B. Heivik 1964 Site and mode of termination of primary vestibulocerebellar fibers in the cat. Arch. ital. Biol., 102: 1-21. Carpenter, M. B., B. M. Stein and P. Peter 1972 Primary vestibulocerebellar fibers in the monkey. Distribution of fibers arising from distinctive cell groups of the vestibular ganglia. Am. J. Anat., 135: 221-250. Carpenter, LM.B., and N. L. Strominger 1964 Cerebellooculomotor fibers in the rhesus monkey. J. Comp. Neur., 123: 211-230. Chan-Palay, V. 1977 Cerebellar Dentate Nucleus. Organization, Cytology and Transmitters. Springer, Berlin, p. 548. Colman, D. R., F. Scalia and E. Cabrales 1976 Light and electron microscopic observations on the anterograde transport of horseradish peroxidase in the optic pathways in the mouse and rat. Brain Res., 102: 156.163. Cowey, A., and C. G. Gross 1970 Effects of foveal prestriate and inferotemporal lesions on visual discrimination by rhesus monkeys. Exp. Brain Res., 11: 128-144. De Olmos. J . S. 1977 An improved HRP method for t h e study of central nervous connections. Exp. Brain Res., 29: 541-551. Flood, S., and J. Jansen 1961 On the cerebellar nuclei in the cat. Acta anat., 46: 52-72. Gacek, R. R. 1977 Location of brain stem neurons projecting to t h e oculomotor nucleus in the cat. Exp. Neurol., 57: 725-749. Garey, L. J., and T. P. S. Powell 1968 The projection of the retina in the cat. J. Anat. (London), 102: 189-222.
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CEREBELLO-PULVINO-CORTICAL PATHWAY IN THE CAT jections from cortical areas 17, 18, and 19 onto the thalamus, pretectum and superior colliculus in the cat. J. Comp. Neur., 173; 81-122. Voogd, J. 1964 The Cerebellum of t h e Cat. Structure and Fibre Connexions. Van Gorcum, Assen, p. 215. Wilson, M., H. M. Kaufman, R. E. Zieler and J. P. Lieb 1972 Visual identifications and memory in monkeys with cir-
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