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Hypothalamic, tectal and accessory optic projections in the opossum
302
Braht Research, 84 (1975) 302 -307 ,~'i~ Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Hypothalamic, tectal and accessory optic projections in the opossum
LENY ALVES CAVALCANTE, CARLOS E D U A R D O R O C H A - M I R A N D A AND ROBERTO LENT
Lab. Neurobiologia 11, Instituto de Biofisica da UFRJ, Centro de Cigncias da Saftde, Bloco G, Cidade Universittlria, 20000 ZC32 Rio de Janeiro (Brazil) (Accepted October 16th, 1974)
Based on experimental studies employing Fink-Heimer methods, Lent and Rocha-Miranda 6 proposed as terminal sites for retinofugal axons in the opossum (Didelphis marsupialis aurita Wied, 1826) the following nuclei: dorsal and ventral lateral geniculate nuclei (GLD and GLV), superior colliculus (CS), nucleus of the optic tract (NTO) and olivary pretectal nucleus (PO) as part of the pretectal complex, and as the only nuclear component of the accessory optic system, the nucleus of the transverse peduncular tract (medial terminal nucleus - - MTN - - in Hayhow's terminology). We have employed radioautographical techniques in order to obtain more detailed information on retinofugal projections in the opossum. The present results refer specifically to hypothalamic, tectal and accessory optic projections. In 3 adult and 5 young opossums under barbiturate anesthesia, 10-20/~Ci of [3H]proline (45.7 Ci/mmole) were injected into the vitreous body under ophthalmoscopic control. The radioactive solution was injected by means of an infusion pump yielding a flux of 1 #l/min. At sacrifice the injected eye was opened and examined under a dissection microscope for evidence of damage. After survival times ranging from 1 to 6 days the animals were deeply anesthetized with pentobarbital and perfused through the aorta with 0.9 ~ NaC1 followed by 4 ~ formaldehyde solution. After perfusion the dorsolateral walls of the skull were removed and fixation was continued by immersion for at least 24 h. Embedding was in paraffin. Each brain was serially sectioned in the coronal plane at 10 #m thickness and every fifth or tenth section was mounted. After removal of paraffin and hydration, sections were prepared for radioautography with Kodak NTB~ emulsion according to the procedure of Kopriwa and Leblond 5 and exposed for periods from 4 to 7 weeks. Development was in Kodak D-19 and fixation in acid fixer. The sections were stained by the Nissl method with cresyl violet. Retinohypothalamicprojections. We have observed the existence of both crossed and uncrossed retinal projections to the suprachiasmatic nucleus (SCh). Silver grains are predominantly found in the posterior part of the nucleus at levels caudal to the
303
Fig. 1. A: schematic drawing of a coronal section of the brain of the opossum at the level where labeling of the suprachiasmatic nucleus (SCh) is maximal. B and C: photomicrographs of the contralateral (B) and ipsilateral (C) SCh in an animal injected intravitreally with 20/~Ci of [SH]proline 5 days before sacrifice. Observe that the density of silver grains in C is about half of that in B. Scale is 50/~m. Abbreviations: SCh, suprachiasmatic nucleus; tro, optic tract; Cd, caudate nucleus; ci, internal capsule; coh, hippocampal commissure; CxA, Ammon cortex; CxC, cingulate cortex; dso, supraoptic decussation; ISTm, interstitial nucleus of the stria terminalis, pars medialis; POM, medial preoptic area; SO, supraoptic nucleus; III, third ventricle; II, lateral ventricle.
304 decussation of the optic fibers, and are about twice as abundant in the contralateral as in the ipsilateral nucleus (Fig. IA-C). Labeled material is already present at 2-day survival time although labeling becomes heavier with longer periods. The ratio of grain densities over the suprachiasmatic nuclei in the two sides is consistent with the observations of Hendrickson et al. 4 in a variety of mammals. The retinohypothalamic projections in the opossum also seem to be confined to the suprachiasmatic nucleus, as cytoarchitectonically defined in this form by Oswaldo-Cruz and Rocha-Miranda-. Our observations lend additional support to Ebbesson's proposal that the retinohypothalamic connection should be considered a component of the basic pattern of retinal projections in vertebrates '~. Superior eollieulus. The crossed projection to the CS presents the same general characteristics as observed with Fink-Heimer methods, i.e., it is distributed mainly to the strata griseum superficiale and zonale. However, radioautographs show a new pattern, not detected in Fink-Heimer sections, of the uncrossed projection which is restricted to the rostral half of the CS. Silver grains appear at the rostralmost region of the ipsilateral colliculus as discrete clusters in the stratum zonale. At more posterior levels, these clusters collect into a narrow band in the mediolateral extent of the superficial third of the stratum zonale. As it extends caudalward the band becomes restricted to more lateral regions. This pattern of ipsilateral grain distribution is complementary to that observed in corresponding parts of the contralateral colliculus (Fig. 2A-C). It could be suspected that this finding represents a small contingent of optic fibers of passage entering the ipsilateral CS superficially. However, it seems unlikely that fibers of passage would be so heavily labeled in the colliculus after only 2 days of axoplasmic transport from the eye. This objection is confirmed by the observation that the stratum opticum is relatively free from silver grains on both sides. Another argument against that interpretation lies in the failure of silver impregnation methods to reveal tangential retinofugal fibers of passage in the stratum zonale. It therefore seems more likely that the complemental localization of silver grains represents a difference in the terminal distribution of crossed and uncrossed retinal fibers. To our knowledge, such a segregation has not been previously described in nonprimate mammals. The pattern of terminal segregation here reported differs from that observed by Tigges and Tigges 9 in Galago erassicaudatus where crossed and un+
Fig. 2. A: schematic drawing of a coronal section of the brain of the opossum at the rostral half of the CS showing also the location of DTN and LTN. B and C: photomicrographs of the upper layers of the CS at the same level as A, contralateral and ipsilateral to the injected eye, respectively. Note that the distribution of silver grains in radioautographs of the 2 sides is roughly complemental. D and E are taken from contralateral DTN and LTN, respectively; the animal had been injected intravitreally with 20/~Ci of [3H]proline, 2 days before sacrifice. Scale is 50/~m. Abbreviations: CS, superior colliculus; sz, zonal layer; sgs, superficial gray layer; so, optic layer; DTN, dorsal terminal nucleus; LTN, lateral terminal nucleus; toa, accessory optic tract; GCd, substantia grisea centralis, pars dorsalis; GM, medial geniculate nucleus; IP, interpeduncular nucleus; TrM, mesencephalic trigeminal nucleus.
305
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306 crossed retinal fibers terminate in different layers of the stratum griseuu~ superB'ciale. However, it is possible that the ipsilateral axon terminals in the opossum synapse with dendrites of cells, the somata of which are in a sublayer of the stratum griseum superfieiale homologous to Galago's substratum B. The laminar segregation we have observed may be of importance for a binocular interaction at collicular levels. Bodian 1 in the Virginia opossum has already described fibers from the extreme temporal retina reaching the rostral ipsilateral CS at the same site where crossed fibers from medial temporal retina appear to terminate. Since in the opossum the optical axes form an angle of about 60°, corresponding points should be located in the extreme temporal retina of one eye and the medial temporal retina of the other. If it is assumed that the extent of the binocular field is an important determinant of binocular interaction, it may well be asked how it is that laminar segregation of retinotectal fiber terminals in the CS has not been observed in other mammalian species with larger binocular visual fields. First, the phenomenon may have escaped detection by conventional anterograde degeneration methods. Furthermore, the increased quantitative dominance of the retinogeniculate over the retinotectal pathway has shifted the relative position of the CS in the visual system throughout the course of evolution of the brain. However, evolution does not necessarily follow a linear course, and it may allow the simultaneous development of complex patterns of anatomical organization in phylogenetically old (CS) and new (GLD) structures, as appears to be the case in Galago. Accessory optic system. In radioautographs the contralateral MTN appears conspicuously labeled and the distribution of silver grains is remarkably similar to that of the terminal degeneration revealed by Fink-Heimer methods 6. No silver grains are found in the ipsilateral nucleus. In addition to the labeling of the MTN, discrete accumulations of grains are observed in regions topographically homologous to the lateral terminal nucleus (LTN) and dorsal terminal nucleus (DTN) of the marsupial phalanger 3 (Fig. 2A, D, E). In the case of the presumable L T N the cluster of grains is situated at the angle between the medial geniculate body and the cerebral peduncle, at a site which has been shown to receive fibers from the upper layers of the ipsilateral CS 8. This region is extremely poor in neuronal perikarya and previous cytoarchitectonic studies of Oswaldo-Cruz and Rocha-Miranda 7 have not allowed a clear-cut characterization of this region as a nucleus. The same remark applies with some qualifications to the cytoarchitectonic characterization of the DTN. Nevertheless, this region is not as sparsely populated as the L T N and radioautography allows the characterization of the DTN as distinct from the pretectal complex. The silver grains are distributed to a region covered by the brachium of the CS and bounded ventrally by the superior pole of the medial geniculate body. At the short survival times employed the accumulation of grains is clearly distinct from the overlying brachium of the CS which appears relatively grain-free. Our findings suggest that the accessory optic system of the opossum follows the generalized plan proposed by Hayhow.
307
We thank Mr. Raymundo Bernardes for valuable technical assistance. This investigation was supported by the Conselho Nacional de Pesquisas (TC No. 16.410), Banco Nacional de Desenvolvimento Econ6mico (FUNTEC 241) and Conselho de Ensino e Pesquisas para Graduados da Universidade Federal do Rio de Janeiro.
1 BODIAN, D., An experimental study of the optic tracts and retinal projection of the Virginia opossum, J. comp. Neurol., 66 (1937) 113-144. 2 EBBESSON,S. O. E., On the organization of central visual pathways in vertebrates, Brain Behav. Evolut., 3 (1970) 178-194. 3 HAYHOW,W. R., The accessory optic system in the marsupial phalanger, Trichosurus vulpecula, J. comp. Neurol., 126 (1966) 653-671. 4 HENDRICKSON, A. E., WAGONER,N., AND COWAN, W. M., An autoradiographic and electron microscopic study of retinohypothalamic connections, Z. Zellforsch., 135 (1972) 1-26. 5 KOPRIWA,B. M., ANDLEBLOND,C. P., Improvements in the coating technique of radioautography, J. Histochem. Cytochem., 10 (1962) 269-284. 6 LENT, R., AND ROCHA-MIRANDA,C. E., Retinal projections in the opossum Didelphis marsupialis aurita, J. comp. Neurol., in press. 70SWALDO-CRuz,E., ANDROCHA=MIRANDA,C. E., The Brain of the Opossum ( Didelphis marsupialis), Instituto de Biofisica da Universidade Federal do Rio de Janeiro, Rio de Janeiro, 1968, 99 pp. 8 RArOLS,J. A., AND MATZKE,H. A., Efferent projections of the superior colliculus in the opossum, J. comp. Neurol., 138 (1970) 147-160. 9 T I ~ , M., AND TI~G~, J., The retinofugal fibers and their terminal nuclei in Galago crassicaudatus (Primates), J. comp. Neurol., 138 (1970) 87-102.
Braht Research, 84 (1975) 302 -307 ,~'i~ Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Hypothalamic, tectal and accessory optic projections in the opossum
LENY ALVES CAVALCANTE, CARLOS E D U A R D O R O C H A - M I R A N D A AND ROBERTO LENT
Lab. Neurobiologia 11, Instituto de Biofisica da UFRJ, Centro de Cigncias da Saftde, Bloco G, Cidade Universittlria, 20000 ZC32 Rio de Janeiro (Brazil) (Accepted October 16th, 1974)
Based on experimental studies employing Fink-Heimer methods, Lent and Rocha-Miranda 6 proposed as terminal sites for retinofugal axons in the opossum (Didelphis marsupialis aurita Wied, 1826) the following nuclei: dorsal and ventral lateral geniculate nuclei (GLD and GLV), superior colliculus (CS), nucleus of the optic tract (NTO) and olivary pretectal nucleus (PO) as part of the pretectal complex, and as the only nuclear component of the accessory optic system, the nucleus of the transverse peduncular tract (medial terminal nucleus - - MTN - - in Hayhow's terminology). We have employed radioautographical techniques in order to obtain more detailed information on retinofugal projections in the opossum. The present results refer specifically to hypothalamic, tectal and accessory optic projections. In 3 adult and 5 young opossums under barbiturate anesthesia, 10-20/~Ci of [3H]proline (45.7 Ci/mmole) were injected into the vitreous body under ophthalmoscopic control. The radioactive solution was injected by means of an infusion pump yielding a flux of 1 #l/min. At sacrifice the injected eye was opened and examined under a dissection microscope for evidence of damage. After survival times ranging from 1 to 6 days the animals were deeply anesthetized with pentobarbital and perfused through the aorta with 0.9 ~ NaC1 followed by 4 ~ formaldehyde solution. After perfusion the dorsolateral walls of the skull were removed and fixation was continued by immersion for at least 24 h. Embedding was in paraffin. Each brain was serially sectioned in the coronal plane at 10 #m thickness and every fifth or tenth section was mounted. After removal of paraffin and hydration, sections were prepared for radioautography with Kodak NTB~ emulsion according to the procedure of Kopriwa and Leblond 5 and exposed for periods from 4 to 7 weeks. Development was in Kodak D-19 and fixation in acid fixer. The sections were stained by the Nissl method with cresyl violet. Retinohypothalamicprojections. We have observed the existence of both crossed and uncrossed retinal projections to the suprachiasmatic nucleus (SCh). Silver grains are predominantly found in the posterior part of the nucleus at levels caudal to the
303
Fig. 1. A: schematic drawing of a coronal section of the brain of the opossum at the level where labeling of the suprachiasmatic nucleus (SCh) is maximal. B and C: photomicrographs of the contralateral (B) and ipsilateral (C) SCh in an animal injected intravitreally with 20/~Ci of [SH]proline 5 days before sacrifice. Observe that the density of silver grains in C is about half of that in B. Scale is 50/~m. Abbreviations: SCh, suprachiasmatic nucleus; tro, optic tract; Cd, caudate nucleus; ci, internal capsule; coh, hippocampal commissure; CxA, Ammon cortex; CxC, cingulate cortex; dso, supraoptic decussation; ISTm, interstitial nucleus of the stria terminalis, pars medialis; POM, medial preoptic area; SO, supraoptic nucleus; III, third ventricle; II, lateral ventricle.
304 decussation of the optic fibers, and are about twice as abundant in the contralateral as in the ipsilateral nucleus (Fig. IA-C). Labeled material is already present at 2-day survival time although labeling becomes heavier with longer periods. The ratio of grain densities over the suprachiasmatic nuclei in the two sides is consistent with the observations of Hendrickson et al. 4 in a variety of mammals. The retinohypothalamic projections in the opossum also seem to be confined to the suprachiasmatic nucleus, as cytoarchitectonically defined in this form by Oswaldo-Cruz and Rocha-Miranda-. Our observations lend additional support to Ebbesson's proposal that the retinohypothalamic connection should be considered a component of the basic pattern of retinal projections in vertebrates '~. Superior eollieulus. The crossed projection to the CS presents the same general characteristics as observed with Fink-Heimer methods, i.e., it is distributed mainly to the strata griseum superficiale and zonale. However, radioautographs show a new pattern, not detected in Fink-Heimer sections, of the uncrossed projection which is restricted to the rostral half of the CS. Silver grains appear at the rostralmost region of the ipsilateral colliculus as discrete clusters in the stratum zonale. At more posterior levels, these clusters collect into a narrow band in the mediolateral extent of the superficial third of the stratum zonale. As it extends caudalward the band becomes restricted to more lateral regions. This pattern of ipsilateral grain distribution is complementary to that observed in corresponding parts of the contralateral colliculus (Fig. 2A-C). It could be suspected that this finding represents a small contingent of optic fibers of passage entering the ipsilateral CS superficially. However, it seems unlikely that fibers of passage would be so heavily labeled in the colliculus after only 2 days of axoplasmic transport from the eye. This objection is confirmed by the observation that the stratum opticum is relatively free from silver grains on both sides. Another argument against that interpretation lies in the failure of silver impregnation methods to reveal tangential retinofugal fibers of passage in the stratum zonale. It therefore seems more likely that the complemental localization of silver grains represents a difference in the terminal distribution of crossed and uncrossed retinal fibers. To our knowledge, such a segregation has not been previously described in nonprimate mammals. The pattern of terminal segregation here reported differs from that observed by Tigges and Tigges 9 in Galago erassicaudatus where crossed and un+
Fig. 2. A: schematic drawing of a coronal section of the brain of the opossum at the rostral half of the CS showing also the location of DTN and LTN. B and C: photomicrographs of the upper layers of the CS at the same level as A, contralateral and ipsilateral to the injected eye, respectively. Note that the distribution of silver grains in radioautographs of the 2 sides is roughly complemental. D and E are taken from contralateral DTN and LTN, respectively; the animal had been injected intravitreally with 20/~Ci of [3H]proline, 2 days before sacrifice. Scale is 50/~m. Abbreviations: CS, superior colliculus; sz, zonal layer; sgs, superficial gray layer; so, optic layer; DTN, dorsal terminal nucleus; LTN, lateral terminal nucleus; toa, accessory optic tract; GCd, substantia grisea centralis, pars dorsalis; GM, medial geniculate nucleus; IP, interpeduncular nucleus; TrM, mesencephalic trigeminal nucleus.
305
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306 crossed retinal fibers terminate in different layers of the stratum griseuu~ superB'ciale. However, it is possible that the ipsilateral axon terminals in the opossum synapse with dendrites of cells, the somata of which are in a sublayer of the stratum griseum superfieiale homologous to Galago's substratum B. The laminar segregation we have observed may be of importance for a binocular interaction at collicular levels. Bodian 1 in the Virginia opossum has already described fibers from the extreme temporal retina reaching the rostral ipsilateral CS at the same site where crossed fibers from medial temporal retina appear to terminate. Since in the opossum the optical axes form an angle of about 60°, corresponding points should be located in the extreme temporal retina of one eye and the medial temporal retina of the other. If it is assumed that the extent of the binocular field is an important determinant of binocular interaction, it may well be asked how it is that laminar segregation of retinotectal fiber terminals in the CS has not been observed in other mammalian species with larger binocular visual fields. First, the phenomenon may have escaped detection by conventional anterograde degeneration methods. Furthermore, the increased quantitative dominance of the retinogeniculate over the retinotectal pathway has shifted the relative position of the CS in the visual system throughout the course of evolution of the brain. However, evolution does not necessarily follow a linear course, and it may allow the simultaneous development of complex patterns of anatomical organization in phylogenetically old (CS) and new (GLD) structures, as appears to be the case in Galago. Accessory optic system. In radioautographs the contralateral MTN appears conspicuously labeled and the distribution of silver grains is remarkably similar to that of the terminal degeneration revealed by Fink-Heimer methods 6. No silver grains are found in the ipsilateral nucleus. In addition to the labeling of the MTN, discrete accumulations of grains are observed in regions topographically homologous to the lateral terminal nucleus (LTN) and dorsal terminal nucleus (DTN) of the marsupial phalanger 3 (Fig. 2A, D, E). In the case of the presumable L T N the cluster of grains is situated at the angle between the medial geniculate body and the cerebral peduncle, at a site which has been shown to receive fibers from the upper layers of the ipsilateral CS 8. This region is extremely poor in neuronal perikarya and previous cytoarchitectonic studies of Oswaldo-Cruz and Rocha-Miranda 7 have not allowed a clear-cut characterization of this region as a nucleus. The same remark applies with some qualifications to the cytoarchitectonic characterization of the DTN. Nevertheless, this region is not as sparsely populated as the L T N and radioautography allows the characterization of the DTN as distinct from the pretectal complex. The silver grains are distributed to a region covered by the brachium of the CS and bounded ventrally by the superior pole of the medial geniculate body. At the short survival times employed the accumulation of grains is clearly distinct from the overlying brachium of the CS which appears relatively grain-free. Our findings suggest that the accessory optic system of the opossum follows the generalized plan proposed by Hayhow.
307
We thank Mr. Raymundo Bernardes for valuable technical assistance. This investigation was supported by the Conselho Nacional de Pesquisas (TC No. 16.410), Banco Nacional de Desenvolvimento Econ6mico (FUNTEC 241) and Conselho de Ensino e Pesquisas para Graduados da Universidade Federal do Rio de Janeiro.
1 BODIAN, D., An experimental study of the optic tracts and retinal projection of the Virginia opossum, J. comp. Neurol., 66 (1937) 113-144. 2 EBBESSON,S. O. E., On the organization of central visual pathways in vertebrates, Brain Behav. Evolut., 3 (1970) 178-194. 3 HAYHOW,W. R., The accessory optic system in the marsupial phalanger, Trichosurus vulpecula, J. comp. Neurol., 126 (1966) 653-671. 4 HENDRICKSON, A. E., WAGONER,N., AND COWAN, W. M., An autoradiographic and electron microscopic study of retinohypothalamic connections, Z. Zellforsch., 135 (1972) 1-26. 5 KOPRIWA,B. M., ANDLEBLOND,C. P., Improvements in the coating technique of radioautography, J. Histochem. Cytochem., 10 (1962) 269-284. 6 LENT, R., AND ROCHA-MIRANDA,C. E., Retinal projections in the opossum Didelphis marsupialis aurita, J. comp. Neurol., in press. 70SWALDO-CRuz,E., ANDROCHA=MIRANDA,C. E., The Brain of the Opossum ( Didelphis marsupialis), Instituto de Biofisica da Universidade Federal do Rio de Janeiro, Rio de Janeiro, 1968, 99 pp. 8 RArOLS,J. A., AND MATZKE,H. A., Efferent projections of the superior colliculus in the opossum, J. comp. Neurol., 138 (1970) 147-160. 9 T I ~ , M., AND TI~G~, J., The retinofugal fibers and their terminal nuclei in Galago crassicaudatus (Primates), J. comp. Neurol., 138 (1970) 87-102.