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Development of retinohypothalamic and accessory optic projections in the opossum
378
Bra#t Research, 144 (1978) 378- 382 ~'3 Elsevier/North-Holland Biomedical Press
Development of retinohypothalamic and accessory optic projections in the opossum
LENY ALVES CAVALCANTE and CARLOS E D U A R D O R O C H A - M I R A N D A Departamento de Neurobiologia, Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Centro de Cidncias da Saitde, Bloco G, Cidade Universitaria, Ilha do Fund~o, 20.000 - - ZC. 32 Rio de Janeiro (Brazil)
(Accepted October 20th, 1977)
The suprachiasmatic nucleus (SCh) and the accessory optic system (AOS) have been proposed as mediators of light action upon circadian rhythms in pineal indole metabolismi8. A close correlation between the onset of degeneration argyrophilia in the SCh of eye-enucleated rats and the establishment of the adrenal rhythm has also been observedL This change in argyrophilia within SCh occurs around eye opening time both in the rat and the hamster 2,14 but is more precocious in the accessory medial terminal nucleus (MTN) of the hamster 14. However, autoradiographic studies in rats have shown an earlier maturation of the retinohypothalamic projection than had been assessed by the reduced silver methods 9,22. The possibility, therefore, remains that through autoradiography the development of retinal projections to SCh and accessory optic nuclei can be shown to be related. The opossum has been chosen as the experimental subject since in this form birth occurs at a relatively immature state 16 and is followed by a protracted course of brain maturation24. Pineal development has been studied in Didelphis azarae ~5. Furthermore, the organization of retinohypothalamic and accessory optic projections has been determined in adult opossums by autoradiographic techniques4,i7,2~. In order to trace the development of these projections, a neutral solution of L-[aH]proline (41 Ci/mmole) was injected into the left eye of pouch-young opossums (Didelphis marsupialis aurita), in quantities commensurate with ocular dimensions (5-75 #Ci in 1-15 #1). The estimated ages 2° and the number of specimens per group, in parenthesis, were: 10 ~ 1 days old (4), 17 ~ 1 days old (2), 23 ~z 2 days old (4), 33 ~ 3 days old (4), 42 :~ 3 days old (2), 52 zL 4 days old (2), 60 zL 4 days old (2). The day of injection for the oldest group occurred at about eye opening time. Survival times for the two younger groups ranged from 20 to 24 hours, while for the older groups usually lasted from 2 to 4 days. Under deep anesthesia, animals in the three youngest groups were decapitated and their heads immersed in Bouin's fluid; for older animals, fixation was initiated by perfusion through the heart with a 4 ~ formaldehyde solution and continued by immersion of the brains in the same fixative. After paraffin embedding, serial coronal sections were cut at thicknesses from 7 to 10/~m,
379
i
Fig. 1. Dark-field photomicrographs of coronal sections through the suprachiasmatic nuclei of opossums injected into one eye with [SH]proline at 17 days (A) and 42 days (B) after birth. Contralateral nucleus to the left. Observe by comparing the relative grain densities of contralateral and ipsilateral nuclei that the uncrossed projection predominates in the younger animals (A), while the reverse is true at later stages (B). Calibration bars: A, 125/~m, B, 225/~m.
according to brain size. Sections were processed for autoradiography following the procedure described by Cowan et al. 7, and either stained with hematoxylin-eosin or cresyl violet. Autoradiographic patterns were compared to those obtained from adult and late pouch-young opossums previously studied 4. Retinohypothalamic projections. In the 10-day-old group, labeled material was evident in both SCh. There was no predominance of grain density on the side contralateral to the injected eye as is found in the mature opossum 4,17,21. In fact, the grain density seemed higher over the ipsilateral nucleus. This slight dominance of the ipsilateral projection became clearer at 17 days (Fig. 1A). Furthermore, grains were scattered up to the dorsal portion of the SCh in both groups. Grain density over this nucleus was about equal in both sides at 23 days and higher on the contralateral side at 33 days, when labeling became virtually restricted to the ventral aspect of the SCh. However, obvious dominance of the contralateral projection was only established at 42 days (Fig. 1B). No additional changes were observed from this age to the time of eye opening (60 days). We may conclude from these observations that the sequence of development of crossed versus uncrossed retinohypothalamic projections may vary in different species. Both autoradiographic and anterograde degeneration data suggested a precedence of the contralateral pathway in the ratZ,9,~2,while a precedence of the ipsilateral projection is suggested by anterograde degeneration studies in the hamster 14. However, the
380
Fig. 2. Medial terminal nucleus (MTN) of opossums injected into the contralateral eye with [3H ]proline at 10 days (A and B), 17 days (C and D) and 23 days (E and F) after birth. Similarly oriented arrowheads in bright-field (A, C and E) and dark-field photomicrographs (B, D and F) point to corresponding regions in adjacent coronal sections. A and B: notice that no distinct cell group corresponds to the accumulation of grains in B. C and D: paucicellular regions faintly outline a group of cells nearly coextensive with the labeled area. E and F: the MTN is well differentiated, while the labeled area extends beyond the nuclear apex. Hematoxylin-eosin staining. Magnification is the same for A-F. Calibration bar =: 120 itm.
d o m i n a n c e o f the c o n t r a l a t e r a l t e r m i n a l field is c o m m o n to a d u l t m a m m a l s 4,8,1~,~7,2 ~,2.,~. The n a t u r e o f the change which is responsible for the v a r i a t i o n o f relative grain densities on the two sides d u r i n g the o p o s s u m n o r m a l d e v e l o p m e n t is u n k n o w n . A p a r t i a l regression o f a quantitatively a n o m a l o u s p r o j e c t i o n to ipsilateral SCh is a plausible hypothesis. It is reasonable to expect t h a t the need for such an e r r o r - c o r r e c t i n g m e c h a n i s m would v a r y in different species. F u r t h e r m o r e , some variability can be expected a m o n g individuals o f the same species, as seems to occur in the d e v e l o p m e n t o f a n o m a l o u s projections f r o m the i s t h m o - o p t i c nucleus to the ipsilateral retina in chicken e m b r y o s 6. O u r o b s e r v a t i o n s d o n o t p e r m i t us to distinguish between a regression o f ipsilateral p r o j e c t i o n s a n d a late acceleration o f the g r o w t h o f t e r m i n a l a r b o r i z a t i o n s o f crossed axons, since o u r d a t a can only be expressed in terms o f ratios between grain densities on the two sides. It should be n o t e d that, at the r o s t r a l pole o f a d u l t o p o s s u m SCh, the grain density
381 over the contralateral nucleus is equal to or even lower than that over the ipsilateral nucleus. Therefore, it could be argued that, in the younger opossums, optic fibers have not yet invaded the caudal levels of the SCh, where the retinal projections are most substantial and predominantly crossed. However, this explanation is not supported by our cytoarchitectonic observations in pouch-young opossums a. Accessory optic projections. In 10-day-old animals, cells of the presumptive MTN are identified within a group adjacent to the medial aspect of the cerebral peduncle. This identification was based on topographical and autoradiographic criteria. The grain density over this area of the ventral tegment is clearly above the background level on the side contralateral to eye injection (Fig. 2A-B). Though the MTN was clearly demarcated by cell-free zones at 17 days, its two cell types19 were not recognized before 23 days. The label within the nucleus did not quite reach its apex at 17 days (Fig. 2C-D), but it extended beyond that at 23 days (Fig. 2E-F). By this time, both cytoarchitectonic and labeling patterns were similar to that of the adult. In animals injected at 10 and 17 days of age, neither lateral nor dorsal terminal nuclei (LTN and DTN) could be identified. The possibility remains that LTN could not be distinguished within the cell mass of the ventral lateral geniculate nucleus (GLV). This is suggested by the finding that the label within the latter extends further caudal than within the dorsal lateral geniculate nucleus5, while the opposite relationships holds for the adult opossum, and by the continuity of GLV and LTN in the rat 10. At 10 and 17 days, it was not possible to distinguish between superior and inferior fascicles of the accessory optic tract. It is unclear whether the late detection of label (at 23 days) in regions topographically homologous to LTN and DTN indicates a late development of the superior fascicle which supplies these nuclei. The labeling of MTN at 10 days argues against this interpretation, since most of its input comes from the superior fascicle la. Furthermore, LTN and DTN are poorly labeled as compared to MTN, even in mature opossums4. Although we have no data on the time the first optic fibers reach the SCh and the MTN, we may conclude that there is no strict correlation between the development of the retinohypothalamic and the accessory optic projections. No additional changes in terminal fields seem to occur for the latter after 23 days, while the adult pattern of retinal projections to the SCh is established between 33 and 42 days. In this connection it is interesting to note that the pineal gland starts developing relatively late in Didelphis. For instance, in Didelphis azarae, formation of the pineal recess starts by the 20th day after birth, and differentiation of the pineal tissue begins only after the first month of life in the pouch 15. This is in sharp contrast with the rat, in which the pineal anlage first appears on the 14th embryonic day and the pineal is relatively well-developed at birth 1, before the first optic fibers reach the SChg,2z. The reason for this discrepancy may be related to differences in the functional importance of the pineal for these species. Didelphis' pineal gland is considered a rudimentary structure on morphological grounds 12,~5, and its functional role with respect to light-controlled activities remains to be determined.
382 We are grateful for the excellent technical assistance of Mr. R a y m u n d o F. Bernardes. This investigation was supported by the Conselho N a c i o n a l de Desenvolvimento Cientifico e Tecnol6gico (TC-16917 a n d TC-2222.0734), by the F i n a n c i a d o r a de Estudos e Projetos ( F N D C T / 3 7 5 - C T ) a n d by the Conselho de Ensino e Pesquisa para G r a d u a d o s of the Universidade Federal do Rio de Janeiro. 1 Ari6ns Kappers, J., The development, topographical relations and innervation of the epiphysis cerebri in the albino rat, Z. Zellforsch., 52 (1960) 163-215. 2 Campbell, C. B. G. and Ramaley, J. A., Retinohypothalamic projections: correlations with onset of the adrenal rhythm in infant rats, Endocrinology, 94 (1974) 1201-1204. 3 Cavalcante, L. A., Desenvolvimento P6s-natal das Pro]e¢6es Retinianas e de Seus Nt~cleos Terminais no Didelphis marsupialis aurita, D.Sc. Thesis, Universidade Federal do Rio de Janeiro, Instituto de Biofisica da UFRJ, Rio de Janeiro, 1976. 4 Cavalcante, L. A., Rocha-Miranda, C. E. and Lent, R., Hypothalamic, tectal and accessory optic projections in the opossum, Brain Research, 84 (1975) 302-307. 5 Cavalcante, L. A. and Rocha-Miranda, C. E., Postnatal development of retinogeniculate, retinopretectal and retinotectal projections in the opossum, Brain Research, in press. 6 Clarke, P. G. H. and Cowan, W. M., The development of the isthmo-optic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons, J. comp. Neurol., 167 (1976) 143-164. 7 Cowan, W. M., Gottlieb, D. I., Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. 8 Eichler, V. B. and Moore, R. Y., The primary and accessory optic systems in the golden hamster, Mesoericetus auratus, Aeta Anat., 89 (1974) 359-371. 9 Felong, M., Development of the retinohypothalamic projection in the rat, Anat. Rec., 184 (1976) 400~01. 10 Hayhow, W. R., Webb, C. and Jervie, A., The accessory optic fiber system in the rat, J. comp. NeuroL, 115 (1960) 187-216. 11 Hendrickson, A. E., Wagoner, N. and Cowan, W. M., An autoradiographic and electron microscopic study of retino-hypothalamic connections, Z. Zellforseh., 135 (1972) 1-26. 12 Jordan, H. E., The microscopic anatomy of the epiphysis of the opossum, Anat. Rec., 5 (1911) 325-338. 13 Lent, R., Cavalcante, L. A. and Rocha-Miranda, C. E., Retinofugal projections in the opossum. An anterograde degeneration and radioautographic study, Brain Research, 107 (1976) 9-26. 14 Leonard, C. M., Degeneration argyrophilia as an index of neural maturation: studies on the optic tract of the golden hamster, J. eomp. NeuroL, 156 (1974) 435-458. 15 Machado, A. B. M., Dados histol6gicos e embriol6gicos sobre a regi~o pineal em alguns marsupiais brasileiros, Ci~neia e Cultura, 17 (1965) 249. 16 McCrady, E., Jr., The Embryology of the Opossum, Amer. Anat. Memoirs, No. 16, Wistar Institute, Philadelphia, 1938, 233 pp. 17 Moore, R. Y., Retinohypothalamicprojections in mammals: a comparative study, Brain Research, 49 (1973) 403-409. 18 Moore, R. Y., Visual pathways and the central neural control of diurnal rhythms. In F. O. Schmitt and F. G. Worden (Eds.), The Neuroscienees 3rd Study Program, MIT Press, Cambridge, 1974, pp. 537-542. 19 Oswaldo-Cruz, E. and Rocha-Miranda, C. E., The Brain of the Opossum ( Didelphis marsupialis), Instituto de Bioflsica da UFRJ, Rio de Janeiro, 1968, 99 pp. 20 Reynolds, H. C., Studies of reproduction in the opossum, Univ. Calif. Publ. Zool., 52 (1952) 233-284. 21 Royce, G. J., Ward, J. P. and Harting, J. K., Retinofugal pathways in two marsupials, J. eomp. Neurol., 170 (1976) 391-414. 22 Stanfield, B. and Cowan, W. M., Evidence for a change in the retino-hypothalamic projection in the rat following early removal of one eye, Brain Research, 104 (1976) 129-136. 23 Thorpe, P., The presence of a retinohypothalamic projection in the ferret, Brain Research, 85 (1975) 343-346. 24 Ulinski, P., External morphology of pouch young oposst m brains: a profile of opossum neurogenesis, J. comp. NeuroL, 142 (1971) 33-58.
Bra#t Research, 144 (1978) 378- 382 ~'3 Elsevier/North-Holland Biomedical Press
Development of retinohypothalamic and accessory optic projections in the opossum
LENY ALVES CAVALCANTE and CARLOS E D U A R D O R O C H A - M I R A N D A Departamento de Neurobiologia, Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Centro de Cidncias da Saitde, Bloco G, Cidade Universitaria, Ilha do Fund~o, 20.000 - - ZC. 32 Rio de Janeiro (Brazil)
(Accepted October 20th, 1977)
The suprachiasmatic nucleus (SCh) and the accessory optic system (AOS) have been proposed as mediators of light action upon circadian rhythms in pineal indole metabolismi8. A close correlation between the onset of degeneration argyrophilia in the SCh of eye-enucleated rats and the establishment of the adrenal rhythm has also been observedL This change in argyrophilia within SCh occurs around eye opening time both in the rat and the hamster 2,14 but is more precocious in the accessory medial terminal nucleus (MTN) of the hamster 14. However, autoradiographic studies in rats have shown an earlier maturation of the retinohypothalamic projection than had been assessed by the reduced silver methods 9,22. The possibility, therefore, remains that through autoradiography the development of retinal projections to SCh and accessory optic nuclei can be shown to be related. The opossum has been chosen as the experimental subject since in this form birth occurs at a relatively immature state 16 and is followed by a protracted course of brain maturation24. Pineal development has been studied in Didelphis azarae ~5. Furthermore, the organization of retinohypothalamic and accessory optic projections has been determined in adult opossums by autoradiographic techniques4,i7,2~. In order to trace the development of these projections, a neutral solution of L-[aH]proline (41 Ci/mmole) was injected into the left eye of pouch-young opossums (Didelphis marsupialis aurita), in quantities commensurate with ocular dimensions (5-75 #Ci in 1-15 #1). The estimated ages 2° and the number of specimens per group, in parenthesis, were: 10 ~ 1 days old (4), 17 ~ 1 days old (2), 23 ~z 2 days old (4), 33 ~ 3 days old (4), 42 :~ 3 days old (2), 52 zL 4 days old (2), 60 zL 4 days old (2). The day of injection for the oldest group occurred at about eye opening time. Survival times for the two younger groups ranged from 20 to 24 hours, while for the older groups usually lasted from 2 to 4 days. Under deep anesthesia, animals in the three youngest groups were decapitated and their heads immersed in Bouin's fluid; for older animals, fixation was initiated by perfusion through the heart with a 4 ~ formaldehyde solution and continued by immersion of the brains in the same fixative. After paraffin embedding, serial coronal sections were cut at thicknesses from 7 to 10/~m,
379
i
Fig. 1. Dark-field photomicrographs of coronal sections through the suprachiasmatic nuclei of opossums injected into one eye with [SH]proline at 17 days (A) and 42 days (B) after birth. Contralateral nucleus to the left. Observe by comparing the relative grain densities of contralateral and ipsilateral nuclei that the uncrossed projection predominates in the younger animals (A), while the reverse is true at later stages (B). Calibration bars: A, 125/~m, B, 225/~m.
according to brain size. Sections were processed for autoradiography following the procedure described by Cowan et al. 7, and either stained with hematoxylin-eosin or cresyl violet. Autoradiographic patterns were compared to those obtained from adult and late pouch-young opossums previously studied 4. Retinohypothalamic projections. In the 10-day-old group, labeled material was evident in both SCh. There was no predominance of grain density on the side contralateral to the injected eye as is found in the mature opossum 4,17,21. In fact, the grain density seemed higher over the ipsilateral nucleus. This slight dominance of the ipsilateral projection became clearer at 17 days (Fig. 1A). Furthermore, grains were scattered up to the dorsal portion of the SCh in both groups. Grain density over this nucleus was about equal in both sides at 23 days and higher on the contralateral side at 33 days, when labeling became virtually restricted to the ventral aspect of the SCh. However, obvious dominance of the contralateral projection was only established at 42 days (Fig. 1B). No additional changes were observed from this age to the time of eye opening (60 days). We may conclude from these observations that the sequence of development of crossed versus uncrossed retinohypothalamic projections may vary in different species. Both autoradiographic and anterograde degeneration data suggested a precedence of the contralateral pathway in the ratZ,9,~2,while a precedence of the ipsilateral projection is suggested by anterograde degeneration studies in the hamster 14. However, the
380
Fig. 2. Medial terminal nucleus (MTN) of opossums injected into the contralateral eye with [3H ]proline at 10 days (A and B), 17 days (C and D) and 23 days (E and F) after birth. Similarly oriented arrowheads in bright-field (A, C and E) and dark-field photomicrographs (B, D and F) point to corresponding regions in adjacent coronal sections. A and B: notice that no distinct cell group corresponds to the accumulation of grains in B. C and D: paucicellular regions faintly outline a group of cells nearly coextensive with the labeled area. E and F: the MTN is well differentiated, while the labeled area extends beyond the nuclear apex. Hematoxylin-eosin staining. Magnification is the same for A-F. Calibration bar =: 120 itm.
d o m i n a n c e o f the c o n t r a l a t e r a l t e r m i n a l field is c o m m o n to a d u l t m a m m a l s 4,8,1~,~7,2 ~,2.,~. The n a t u r e o f the change which is responsible for the v a r i a t i o n o f relative grain densities on the two sides d u r i n g the o p o s s u m n o r m a l d e v e l o p m e n t is u n k n o w n . A p a r t i a l regression o f a quantitatively a n o m a l o u s p r o j e c t i o n to ipsilateral SCh is a plausible hypothesis. It is reasonable to expect t h a t the need for such an e r r o r - c o r r e c t i n g m e c h a n i s m would v a r y in different species. F u r t h e r m o r e , some variability can be expected a m o n g individuals o f the same species, as seems to occur in the d e v e l o p m e n t o f a n o m a l o u s projections f r o m the i s t h m o - o p t i c nucleus to the ipsilateral retina in chicken e m b r y o s 6. O u r o b s e r v a t i o n s d o n o t p e r m i t us to distinguish between a regression o f ipsilateral p r o j e c t i o n s a n d a late acceleration o f the g r o w t h o f t e r m i n a l a r b o r i z a t i o n s o f crossed axons, since o u r d a t a can only be expressed in terms o f ratios between grain densities on the two sides. It should be n o t e d that, at the r o s t r a l pole o f a d u l t o p o s s u m SCh, the grain density
381 over the contralateral nucleus is equal to or even lower than that over the ipsilateral nucleus. Therefore, it could be argued that, in the younger opossums, optic fibers have not yet invaded the caudal levels of the SCh, where the retinal projections are most substantial and predominantly crossed. However, this explanation is not supported by our cytoarchitectonic observations in pouch-young opossums a. Accessory optic projections. In 10-day-old animals, cells of the presumptive MTN are identified within a group adjacent to the medial aspect of the cerebral peduncle. This identification was based on topographical and autoradiographic criteria. The grain density over this area of the ventral tegment is clearly above the background level on the side contralateral to eye injection (Fig. 2A-B). Though the MTN was clearly demarcated by cell-free zones at 17 days, its two cell types19 were not recognized before 23 days. The label within the nucleus did not quite reach its apex at 17 days (Fig. 2C-D), but it extended beyond that at 23 days (Fig. 2E-F). By this time, both cytoarchitectonic and labeling patterns were similar to that of the adult. In animals injected at 10 and 17 days of age, neither lateral nor dorsal terminal nuclei (LTN and DTN) could be identified. The possibility remains that LTN could not be distinguished within the cell mass of the ventral lateral geniculate nucleus (GLV). This is suggested by the finding that the label within the latter extends further caudal than within the dorsal lateral geniculate nucleus5, while the opposite relationships holds for the adult opossum, and by the continuity of GLV and LTN in the rat 10. At 10 and 17 days, it was not possible to distinguish between superior and inferior fascicles of the accessory optic tract. It is unclear whether the late detection of label (at 23 days) in regions topographically homologous to LTN and DTN indicates a late development of the superior fascicle which supplies these nuclei. The labeling of MTN at 10 days argues against this interpretation, since most of its input comes from the superior fascicle la. Furthermore, LTN and DTN are poorly labeled as compared to MTN, even in mature opossums4. Although we have no data on the time the first optic fibers reach the SCh and the MTN, we may conclude that there is no strict correlation between the development of the retinohypothalamic and the accessory optic projections. No additional changes in terminal fields seem to occur for the latter after 23 days, while the adult pattern of retinal projections to the SCh is established between 33 and 42 days. In this connection it is interesting to note that the pineal gland starts developing relatively late in Didelphis. For instance, in Didelphis azarae, formation of the pineal recess starts by the 20th day after birth, and differentiation of the pineal tissue begins only after the first month of life in the pouch 15. This is in sharp contrast with the rat, in which the pineal anlage first appears on the 14th embryonic day and the pineal is relatively well-developed at birth 1, before the first optic fibers reach the SChg,2z. The reason for this discrepancy may be related to differences in the functional importance of the pineal for these species. Didelphis' pineal gland is considered a rudimentary structure on morphological grounds 12,~5, and its functional role with respect to light-controlled activities remains to be determined.
382 We are grateful for the excellent technical assistance of Mr. R a y m u n d o F. Bernardes. This investigation was supported by the Conselho N a c i o n a l de Desenvolvimento Cientifico e Tecnol6gico (TC-16917 a n d TC-2222.0734), by the F i n a n c i a d o r a de Estudos e Projetos ( F N D C T / 3 7 5 - C T ) a n d by the Conselho de Ensino e Pesquisa para G r a d u a d o s of the Universidade Federal do Rio de Janeiro. 1 Ari6ns Kappers, J., The development, topographical relations and innervation of the epiphysis cerebri in the albino rat, Z. Zellforsch., 52 (1960) 163-215. 2 Campbell, C. B. G. and Ramaley, J. A., Retinohypothalamic projections: correlations with onset of the adrenal rhythm in infant rats, Endocrinology, 94 (1974) 1201-1204. 3 Cavalcante, L. A., Desenvolvimento P6s-natal das Pro]e¢6es Retinianas e de Seus Nt~cleos Terminais no Didelphis marsupialis aurita, D.Sc. Thesis, Universidade Federal do Rio de Janeiro, Instituto de Biofisica da UFRJ, Rio de Janeiro, 1976. 4 Cavalcante, L. A., Rocha-Miranda, C. E. and Lent, R., Hypothalamic, tectal and accessory optic projections in the opossum, Brain Research, 84 (1975) 302-307. 5 Cavalcante, L. A. and Rocha-Miranda, C. E., Postnatal development of retinogeniculate, retinopretectal and retinotectal projections in the opossum, Brain Research, in press. 6 Clarke, P. G. H. and Cowan, W. M., The development of the isthmo-optic tract in the chick, with special reference to the occurrence and correction of developmental errors in the location and connections of isthmo-optic neurons, J. comp. Neurol., 167 (1976) 143-164. 7 Cowan, W. M., Gottlieb, D. I., Hendrickson, A. E., Price, J. L. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. 8 Eichler, V. B. and Moore, R. Y., The primary and accessory optic systems in the golden hamster, Mesoericetus auratus, Aeta Anat., 89 (1974) 359-371. 9 Felong, M., Development of the retinohypothalamic projection in the rat, Anat. Rec., 184 (1976) 400~01. 10 Hayhow, W. R., Webb, C. and Jervie, A., The accessory optic fiber system in the rat, J. comp. NeuroL, 115 (1960) 187-216. 11 Hendrickson, A. E., Wagoner, N. and Cowan, W. M., An autoradiographic and electron microscopic study of retino-hypothalamic connections, Z. Zellforseh., 135 (1972) 1-26. 12 Jordan, H. E., The microscopic anatomy of the epiphysis of the opossum, Anat. Rec., 5 (1911) 325-338. 13 Lent, R., Cavalcante, L. A. and Rocha-Miranda, C. E., Retinofugal projections in the opossum. An anterograde degeneration and radioautographic study, Brain Research, 107 (1976) 9-26. 14 Leonard, C. M., Degeneration argyrophilia as an index of neural maturation: studies on the optic tract of the golden hamster, J. eomp. NeuroL, 156 (1974) 435-458. 15 Machado, A. B. M., Dados histol6gicos e embriol6gicos sobre a regi~o pineal em alguns marsupiais brasileiros, Ci~neia e Cultura, 17 (1965) 249. 16 McCrady, E., Jr., The Embryology of the Opossum, Amer. Anat. Memoirs, No. 16, Wistar Institute, Philadelphia, 1938, 233 pp. 17 Moore, R. Y., Retinohypothalamicprojections in mammals: a comparative study, Brain Research, 49 (1973) 403-409. 18 Moore, R. Y., Visual pathways and the central neural control of diurnal rhythms. In F. O. Schmitt and F. G. Worden (Eds.), The Neuroscienees 3rd Study Program, MIT Press, Cambridge, 1974, pp. 537-542. 19 Oswaldo-Cruz, E. and Rocha-Miranda, C. E., The Brain of the Opossum ( Didelphis marsupialis), Instituto de Bioflsica da UFRJ, Rio de Janeiro, 1968, 99 pp. 20 Reynolds, H. C., Studies of reproduction in the opossum, Univ. Calif. Publ. Zool., 52 (1952) 233-284. 21 Royce, G. J., Ward, J. P. and Harting, J. K., Retinofugal pathways in two marsupials, J. eomp. Neurol., 170 (1976) 391-414. 22 Stanfield, B. and Cowan, W. M., Evidence for a change in the retino-hypothalamic projection in the rat following early removal of one eye, Brain Research, 104 (1976) 129-136. 23 Thorpe, P., The presence of a retinohypothalamic projection in the ferret, Brain Research, 85 (1975) 343-346. 24 Ulinski, P., External morphology of pouch young oposst m brains: a profile of opossum neurogenesis, J. comp. NeuroL, 142 (1971) 33-58.