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The suprachiasmatic nuclei and retinohypothalamic tract in the western spotted skunk
Brain Research, 339 (1985) 378-381
378
Elsevier BRE 20960
The suprachiasmatic nuclei and retinohypothalamic tract in the western spotted skunk RON MAY, MARK DeSANTIS and RODNEY A. MEAD
Department of BiologicalSciences, Universityof Idaho, Moscow, ID 83843 (U.S.A.) (Accepted March 12th, 1985)
Key words: spotted skunk - - suprachiasmatic nucleus - - retinohypothalamic tract
The western spotted skunk has a well-developed retinohypothalamic tract projecting to the middle and caudal parts of the suprachiasmatic nuclei. The nuclei have a complex 3-dimensional shape and contain small neuronal somas. The western spotted skunk has a p r o l o n g e d p e r i o d of embryonic diapause lasting about 200-220 days during which the blastocysts remain unimplanted. R e n e w e d embryonic d e v e l o p m e n t and implantation are triggered by increasing daylength and occur in A p r i l when p h o t o p e r i o d exceeds 14 h of light per dayS,9. In m a m m a l s the suprachiasmatic nuclei (SCN) are responsible for mediating changes associated with p h o t o p e r i o d i c stimuli H. Circadian rhythms of electrical and metabolic activity occur in the SCN of rats 4-6,t2,14. Lesions of the SCN modify photoperiodic induction of reproductive activity in hamsters and rats1,2,13,17 and electrical stimulation of the SCN produces a phase shift in circadian rhythms 16. Changes in daylength are thought to influence the SCN via the r e t i n o h y p o t h a l a m i c tract ( R H T ) , which has b e e n d e m o n s t r a t e d in a n u m b e r of mammals 10 including the ferret 19. The objectives of this study were to determine: (1) the size and 3-dimensional shape of the SCN, (2) the size of cells forming the SCN and (3) the distribution of the R H T within the SCN of the spotted skunk. F e m a l e spotted skunks (Spilogale putorius latifrons) were o b t a i n e d from a t r a p p e r in O r e g o n and maintained as previously described 9. Ten skunks were anesthetized with p e n t o b a r b i t a l (40 mg/kg, i.p.) and perfused with either 10% f o r m a l d e h y d e or Bouin's fixative. Frozen sections of the brain were cut in a coronal (n = 8), horizontal (n = 1), or sagittal
(n = 1) plane and stained with thionin3. Sections (75 /zm thick) containing the SCN were p r o j e c t e d and traced. Dimensions of the SCN were o b t a i n e d from the tracings and a 3-dimensional m o d e l was constructed. The shortest dimension of 150 r a n d o m l y selected cells in which a nucleus was visible was measured throughout the a n t e r i o r - p o s t e r i o r extent of the SCN. O t h e r h y p o t h a l a m i c neurons were similarly measured using a c a m e r a lucida in conjunction with a computer system ( A p p l e IIe, B i o q u a n t software). Values are p r e s e n t e d as the m e a n + the standard deviation. A N O V A and D u n c a n ' s Multiple R a n g e Test were used to test for statistical differences in size among cell groups.
Fig. 1. Three-dimensional drawing of the SCN in the western spotted skunk. The SCN (stippled) is dorsal to the optic nerves, chiasm, and tracts (clear). Sections nearer the foreground are more rostral. Note the dorsoventral elongation of the SCN and convergence of each SCN toward midline as one looks from rostral to caudal.
Correspondence: R. A. Mead, Department of Biological Sciences, University of Idaho, Moscow, ID 83843, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
379
Fig. 2. Photomicrographs of sections through various levels of the skunk's SCN. Darkfield photomicrographs at A, C and E are autoradiographs of coronal sections taken from the rostral, middle and caudal parts of the SCN, respectively. Arrowheads point to silver grains over RHT fibers distributing in the middle and caudal parts of the SCN. Dotted lines mark the boundaries of the SCN in corrcsponding levels of thionin-stained histological sections 475 (B), 775 (D) and 1075 (F) ~m behind the rostral border of the optic chiasm (OC). Arrows indicate the position of the SCN in parasagittal (G) and horizontal (H) sections.
380 To study the retinohypothalamic tract (RHT), the vitreous humor of the right eye was injected with a 3H-L-amino acid mixture (20 btCi, New England Nuclear, NET-250 containing alanine, arginine, aspartate, glutamate, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine, valine) in 10 ~tl of sterile saline. Skunks were anesthetized, perfused with Bouin's solution at 7 days (n = 5) postinjection and the brains embedded in paraffin and sectioned at 10 itm. Two sections were taken at every 100 u m interval and placed in sequence on separate sets of slides. One set was processed for autoradiography~S and stained with thionin. The other set was stained with thionin only. It was not possible to delineate the boundaries of the SCN clearly in these 10 ktm sections which made it difficult to study the exact location of R H T fibers in the SCN. This problem was overcome by superimposing the projection image of the autoradiograph onto appropriate tracings from the 75 g m thick frozen sections. Sections were considered 1o be at about the same level of the hypothalamus when the width of the optic chiasm and the distance posterior to the initial junction of the optic nerves matched between the sections. The SCN of the western spotted skunk was present bilaterally. Starting at 515 + 89/~m behind the anterior border of the optic chiasm it was situated immediately dorsal to the optic chiasm and just ventrolateral to the third ventricle (Fig. 1). The SCN measured 1175 + 114 ~m in rostrocaudal length, attained maximum height of 1046 _+ 77 u m near the caudal end of the nucleus, and reached a maximum width of 799 _+ 153/~m about midway along the length of the nucleus (Fig. 1). The shortest dimension of cells within the SCN was 9.6 _+ 2.0 ktm. In contrast, cells immediately above the SCN had a minimum dimension of 14.9 _+_3.51tm and perikarya of the magnocellular neurons in the su-
praoptic nuclei averaged 24.0 _+ 4.3/ml. Cells in cacti of these areas were significantly different in size from each other (P < 0.05). Fibers of the R H T were located in the medial and ventral region of the SCN. Along the rostro-caudal axis, fibers extended from the middle to the caudal end of each nucleus (Fig. 2A, C, E). The density of silver grains over the contra- and ipsilaterat nuclei was similar indicating that afferent fibers from the right retina were distributed to both SCN about equally. This is the first description of the mustelid SCN. When compared with species examined previously 7,20, the SCN of the western spotted skunk most closely resembles that of the cat. In these species of Carnivora, the posterior poles of the SCN converge toward the midline and are elongated dorsoventrally, which places~their dorsal boundary near the ventral aspect of the paraventricular nuclei. In contrast, the 3-dimensional shape of the SCN of rodents is less complex 7. Although still quite small, the average cellular size in the skunk's SCN (9.6/,m) is somewhat larger than that of the rat (7.83,um), mouse (7.0 jLm) and hamster (7.85 ~m). The distribution of labeled fibers within the skunk's SCN 7 days after injection documents the existence of the R H T in this animal. Labeled fibers in the hypothalamus were confined to the caudal two-thirds of the SCN which suggests that the R H T terminated within the SCN rather than passing through it to other areas. I'he western spotted skunk like the hamster, chimpanzee, and macaque 18has an equal bilateral retinal innervation of the SCN. In several other species the SCN receives a greater input from the contralateral retina~.~.
1 Bittman, E. L., Goldman, B. D. and Zucker, I., Testicular responses to melatonin are altered by lesions of the suprachiasmatic nuclei in golden hamsters, Biol. Reprod., 21 (1979) 647-656. 2 Brown-Grant, K. and Raisman, G., Abnormalities in reproductive function associated with the destruction of the suprachiasmatic nuclei in female rats, Proc. roy. Soc. B, 198 (1977) 279- 296. 3 Clark, G., Tissue preparation and basic staining tech-
niques. In R. T. Robertson (Ed.), Neuroanatomical Research Techniques, Academic Press, New York, 1978, pp.
We thank Heidi Mead for preparing the illustrations. This study was partially supported by grant H D 06556 from the National Institute of Child Health and H u m a n Development.
36-37. 4 Groos, G. A. and Hendriks, J., Regularly firing neurones in the rat suprachiasmatic nucleus, Experientia. 35 (1979) 1597-1598. 5 Groos, G. and Hendriks, J., Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro, Neurosci. Lett., 34 (1982) 283-288.
381 6 Inouye, S. T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic nucleus, J. comp. Physiol., 146 (1982) 153-160. 7 Lydic, R., Albers, H. E., Tepper, B. and Moore-Ede, M. C., Three-dimensional structure of the mammalian suprachiasmatic nuclei: a comparative study of five species, J. comp. Neurol., 204 (1982) 225-237. 8 Mead, R. A., Reproduction in western forms of the spotted skunk (Genus Spilogale), J. Mammal., 49 (1968) 373-390. 9 Mead, R. A., Effects of light and blinding upon delayed implantation in the spotted skunk, Biol. Reprod., 5 (1971) 214-22(I. 10 Moore, R. Y., Retinohypothalamic projection in mammals: a comparative study, Brain Research, 49 (19731 403-409. 11 Moore, R. Y., Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783-2789. 12 Ono, T., Nishino, H., Saski, K., Fukuda, M. and Muramoto, K., Long-term lateral hypothalamic single unit analysis and feeding behavior in freely moving rats, Neurosci. Lett., 26 (19811 79-83. 13 Raisman, G. and Brown-Grant, K., The "suprachiasmatic
14
15 16
17
18
19 20
syndrome': endocrine and behavioural abnormalities following lesions of the suprachiasmatic nuclei in the female rat, Proc. roy. Soc. B, 198 (1977) 297-314. Reppert, S. M. and Schwartz, W. J., The suprachiasmatic nuclei of the fetal rat: characterization of a functional circadian clock using 14C-labeled deoxyglucose, J. Neurosci., 4 (1984) 1677-1682. Rogers, A. W., Techniques of Autoradiography, Elsevier, Amsterdam, 1969, pp. 253-272. Rusak. B. and Groos, G., Suprachiasmatic stimulation phase shifts rodent circadian rhythms, Science, 215 (1982) 1407-1409. Rusak, B. and Morin, L. P., Testicular responses to photoperiod are blocked by lesions of the suprachiasmatic nuclei in golden hamsters, Biol. Reprod., 15 (19761 366-374. Silvermam A. and Pickard, G., The hypothalamus. In P. C. Emson (Ed.), Chemical Neuroanatomy, Raven Press, New York, 1983, pp. 316-320. Thorpe, P. A., The presence of a retinohypothalamic projection in the ferret, Brain Research, 85 (1975) 343-346. Van den Pol, A. N., The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy, J. comp. Neurol., 191 (1980) 661-702.
378
Elsevier BRE 20960
The suprachiasmatic nuclei and retinohypothalamic tract in the western spotted skunk RON MAY, MARK DeSANTIS and RODNEY A. MEAD
Department of BiologicalSciences, Universityof Idaho, Moscow, ID 83843 (U.S.A.) (Accepted March 12th, 1985)
Key words: spotted skunk - - suprachiasmatic nucleus - - retinohypothalamic tract
The western spotted skunk has a well-developed retinohypothalamic tract projecting to the middle and caudal parts of the suprachiasmatic nuclei. The nuclei have a complex 3-dimensional shape and contain small neuronal somas. The western spotted skunk has a p r o l o n g e d p e r i o d of embryonic diapause lasting about 200-220 days during which the blastocysts remain unimplanted. R e n e w e d embryonic d e v e l o p m e n t and implantation are triggered by increasing daylength and occur in A p r i l when p h o t o p e r i o d exceeds 14 h of light per dayS,9. In m a m m a l s the suprachiasmatic nuclei (SCN) are responsible for mediating changes associated with p h o t o p e r i o d i c stimuli H. Circadian rhythms of electrical and metabolic activity occur in the SCN of rats 4-6,t2,14. Lesions of the SCN modify photoperiodic induction of reproductive activity in hamsters and rats1,2,13,17 and electrical stimulation of the SCN produces a phase shift in circadian rhythms 16. Changes in daylength are thought to influence the SCN via the r e t i n o h y p o t h a l a m i c tract ( R H T ) , which has b e e n d e m o n s t r a t e d in a n u m b e r of mammals 10 including the ferret 19. The objectives of this study were to determine: (1) the size and 3-dimensional shape of the SCN, (2) the size of cells forming the SCN and (3) the distribution of the R H T within the SCN of the spotted skunk. F e m a l e spotted skunks (Spilogale putorius latifrons) were o b t a i n e d from a t r a p p e r in O r e g o n and maintained as previously described 9. Ten skunks were anesthetized with p e n t o b a r b i t a l (40 mg/kg, i.p.) and perfused with either 10% f o r m a l d e h y d e or Bouin's fixative. Frozen sections of the brain were cut in a coronal (n = 8), horizontal (n = 1), or sagittal
(n = 1) plane and stained with thionin3. Sections (75 /zm thick) containing the SCN were p r o j e c t e d and traced. Dimensions of the SCN were o b t a i n e d from the tracings and a 3-dimensional m o d e l was constructed. The shortest dimension of 150 r a n d o m l y selected cells in which a nucleus was visible was measured throughout the a n t e r i o r - p o s t e r i o r extent of the SCN. O t h e r h y p o t h a l a m i c neurons were similarly measured using a c a m e r a lucida in conjunction with a computer system ( A p p l e IIe, B i o q u a n t software). Values are p r e s e n t e d as the m e a n + the standard deviation. A N O V A and D u n c a n ' s Multiple R a n g e Test were used to test for statistical differences in size among cell groups.
Fig. 1. Three-dimensional drawing of the SCN in the western spotted skunk. The SCN (stippled) is dorsal to the optic nerves, chiasm, and tracts (clear). Sections nearer the foreground are more rostral. Note the dorsoventral elongation of the SCN and convergence of each SCN toward midline as one looks from rostral to caudal.
Correspondence: R. A. Mead, Department of Biological Sciences, University of Idaho, Moscow, ID 83843, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
379
Fig. 2. Photomicrographs of sections through various levels of the skunk's SCN. Darkfield photomicrographs at A, C and E are autoradiographs of coronal sections taken from the rostral, middle and caudal parts of the SCN, respectively. Arrowheads point to silver grains over RHT fibers distributing in the middle and caudal parts of the SCN. Dotted lines mark the boundaries of the SCN in corrcsponding levels of thionin-stained histological sections 475 (B), 775 (D) and 1075 (F) ~m behind the rostral border of the optic chiasm (OC). Arrows indicate the position of the SCN in parasagittal (G) and horizontal (H) sections.
380 To study the retinohypothalamic tract (RHT), the vitreous humor of the right eye was injected with a 3H-L-amino acid mixture (20 btCi, New England Nuclear, NET-250 containing alanine, arginine, aspartate, glutamate, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine, valine) in 10 ~tl of sterile saline. Skunks were anesthetized, perfused with Bouin's solution at 7 days (n = 5) postinjection and the brains embedded in paraffin and sectioned at 10 itm. Two sections were taken at every 100 u m interval and placed in sequence on separate sets of slides. One set was processed for autoradiography~S and stained with thionin. The other set was stained with thionin only. It was not possible to delineate the boundaries of the SCN clearly in these 10 ktm sections which made it difficult to study the exact location of R H T fibers in the SCN. This problem was overcome by superimposing the projection image of the autoradiograph onto appropriate tracings from the 75 g m thick frozen sections. Sections were considered 1o be at about the same level of the hypothalamus when the width of the optic chiasm and the distance posterior to the initial junction of the optic nerves matched between the sections. The SCN of the western spotted skunk was present bilaterally. Starting at 515 + 89/~m behind the anterior border of the optic chiasm it was situated immediately dorsal to the optic chiasm and just ventrolateral to the third ventricle (Fig. 1). The SCN measured 1175 + 114 ~m in rostrocaudal length, attained maximum height of 1046 _+ 77 u m near the caudal end of the nucleus, and reached a maximum width of 799 _+ 153/~m about midway along the length of the nucleus (Fig. 1). The shortest dimension of cells within the SCN was 9.6 _+ 2.0 ktm. In contrast, cells immediately above the SCN had a minimum dimension of 14.9 _+_3.51tm and perikarya of the magnocellular neurons in the su-
praoptic nuclei averaged 24.0 _+ 4.3/ml. Cells in cacti of these areas were significantly different in size from each other (P < 0.05). Fibers of the R H T were located in the medial and ventral region of the SCN. Along the rostro-caudal axis, fibers extended from the middle to the caudal end of each nucleus (Fig. 2A, C, E). The density of silver grains over the contra- and ipsilaterat nuclei was similar indicating that afferent fibers from the right retina were distributed to both SCN about equally. This is the first description of the mustelid SCN. When compared with species examined previously 7,20, the SCN of the western spotted skunk most closely resembles that of the cat. In these species of Carnivora, the posterior poles of the SCN converge toward the midline and are elongated dorsoventrally, which places~their dorsal boundary near the ventral aspect of the paraventricular nuclei. In contrast, the 3-dimensional shape of the SCN of rodents is less complex 7. Although still quite small, the average cellular size in the skunk's SCN (9.6/,m) is somewhat larger than that of the rat (7.83,um), mouse (7.0 jLm) and hamster (7.85 ~m). The distribution of labeled fibers within the skunk's SCN 7 days after injection documents the existence of the R H T in this animal. Labeled fibers in the hypothalamus were confined to the caudal two-thirds of the SCN which suggests that the R H T terminated within the SCN rather than passing through it to other areas. I'he western spotted skunk like the hamster, chimpanzee, and macaque 18has an equal bilateral retinal innervation of the SCN. In several other species the SCN receives a greater input from the contralateral retina~.~.
1 Bittman, E. L., Goldman, B. D. and Zucker, I., Testicular responses to melatonin are altered by lesions of the suprachiasmatic nuclei in golden hamsters, Biol. Reprod., 21 (1979) 647-656. 2 Brown-Grant, K. and Raisman, G., Abnormalities in reproductive function associated with the destruction of the suprachiasmatic nuclei in female rats, Proc. roy. Soc. B, 198 (1977) 279- 296. 3 Clark, G., Tissue preparation and basic staining tech-
niques. In R. T. Robertson (Ed.), Neuroanatomical Research Techniques, Academic Press, New York, 1978, pp.
We thank Heidi Mead for preparing the illustrations. This study was partially supported by grant H D 06556 from the National Institute of Child Health and H u m a n Development.
36-37. 4 Groos, G. A. and Hendriks, J., Regularly firing neurones in the rat suprachiasmatic nucleus, Experientia. 35 (1979) 1597-1598. 5 Groos, G. and Hendriks, J., Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro, Neurosci. Lett., 34 (1982) 283-288.
381 6 Inouye, S. T. and Kawamura, H., Characteristics of a circadian pacemaker in the suprachiasmatic nucleus, J. comp. Physiol., 146 (1982) 153-160. 7 Lydic, R., Albers, H. E., Tepper, B. and Moore-Ede, M. C., Three-dimensional structure of the mammalian suprachiasmatic nuclei: a comparative study of five species, J. comp. Neurol., 204 (1982) 225-237. 8 Mead, R. A., Reproduction in western forms of the spotted skunk (Genus Spilogale), J. Mammal., 49 (1968) 373-390. 9 Mead, R. A., Effects of light and blinding upon delayed implantation in the spotted skunk, Biol. Reprod., 5 (1971) 214-22(I. 10 Moore, R. Y., Retinohypothalamic projection in mammals: a comparative study, Brain Research, 49 (19731 403-409. 11 Moore, R. Y., Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783-2789. 12 Ono, T., Nishino, H., Saski, K., Fukuda, M. and Muramoto, K., Long-term lateral hypothalamic single unit analysis and feeding behavior in freely moving rats, Neurosci. Lett., 26 (19811 79-83. 13 Raisman, G. and Brown-Grant, K., The "suprachiasmatic
14
15 16
17
18
19 20
syndrome': endocrine and behavioural abnormalities following lesions of the suprachiasmatic nuclei in the female rat, Proc. roy. Soc. B, 198 (1977) 297-314. Reppert, S. M. and Schwartz, W. J., The suprachiasmatic nuclei of the fetal rat: characterization of a functional circadian clock using 14C-labeled deoxyglucose, J. Neurosci., 4 (1984) 1677-1682. Rogers, A. W., Techniques of Autoradiography, Elsevier, Amsterdam, 1969, pp. 253-272. Rusak. B. and Groos, G., Suprachiasmatic stimulation phase shifts rodent circadian rhythms, Science, 215 (1982) 1407-1409. Rusak, B. and Morin, L. P., Testicular responses to photoperiod are blocked by lesions of the suprachiasmatic nuclei in golden hamsters, Biol. Reprod., 15 (19761 366-374. Silvermam A. and Pickard, G., The hypothalamus. In P. C. Emson (Ed.), Chemical Neuroanatomy, Raven Press, New York, 1983, pp. 316-320. Thorpe, P. A., The presence of a retinohypothalamic projection in the ferret, Brain Research, 85 (1975) 343-346. Van den Pol, A. N., The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy, J. comp. Neurol., 191 (1980) 661-702.