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Thyroxine influences neuronal connectivity in the adult frog brain
Brain Research, 492 (1989) 389-391 Elsevier
389
BRE 23608
Thyroxine influences neuronal connectivity in the adult frog brain M.H. Hofmann, A. Michler and D.L. Meyer Department of Anatomy, School of Medicine, Universityof Goettingen, Goettingen (E R. G.) (Accepted 28 March 1989) Key words: Thyroxine; Visual system; Optic tectum; Neuronal sprouting; Neuronal plasticity; Amphibian
In contrast to results of earlier investigations the influence of thyroxine on CNS connectivity is not restricted to circum-metamorphic stages in frogs. Neuroanatomical findings in adult Xenopus treated with thyroxine reveal a spread of the ipsilaterai retino-tectal projection. Sprouting fibers establish a tectal innervation pattern similar to the one found in primitive fish. The question arises, whether thyroxine also has morphogenetic effects in the mature CNS of other species. It has been established that thyroxine influences changes in CNS connectivity, particularly in the visual system, during metamorphosis in amphibians 1' 13. Hoskinsl0 and Hoskins and Grobstein 11'12 demonstrated that the ipsilateral retino-thalamic projection develops, if low dosages of thyroxine are applied to one retina of a tadpole. The local administration of thyroxine did not result in serum levels required to induce metamorphosis, or the rostrally directed translocation of orbits and eyes. These elegant experiments prove that thyroxine does not change the anatomy of the skull until an enlarged binocular visual field has been created, which then might serve as a trigger for the development of uncrossed visual projections. Hence, it can be concluded that thyroxine is directly involved in the growth of some retinal axons that terminate in the ipsilateral half of the brain. Although a variety of observations of thyroxine effects on the developing amphibian brain has been reported, detailed studies of possible morphogenetic effects of this hormone in adult animals are lacking or have led to negative results. It still is generally accepted that the significance of thyroxine for CNS connectivity is limited to the critical period of metamorphosis s, whereas in adult specimens its role appears to be limited to the control of metabolism 9. This assumption is partly based on changes of
thyroxine production levels in amphibians during different developmental stages. Thyroid activity is low until shortly before metamorphosis, then increases during metamorphosis, and rapidly drops to approximately premetamorphic levels after the tadpole has become a frog 6'17. This has led to the conclusion that thyroxine loses much of its significance for the organism once metamorphosis has been completed. To investigate possible effects of thyroxine on the internal morphology of an adult vertebrate brain we injected 8 adult Xenopus of both sexes and 30-40 g b. wt. with a daily dose of 50 ~g of this hormone for 30 days. The thyroxine was dissolved in 0.125 ml S6rensen buffer (0.05 M, pH 7.4) and injected into the dorsal lymphatic sac. The specimens were kept at 24 °C in an aquarium providing a seminatural environment. After the 30 days of treatment, we performed a tracer study using conventional horseradish peroxidase (HRP) techniques 2'14 with a 3-day survival time after unilateral intraocular HRP injection. During this 3-day period, daily thyroxine applications were continued. As during previous intraocular HRP injections (5 /~l of a 30% solution of HRP dissolved in phosphate buffer containing 1% DMSO, 1% lysolecithin, and 1% kainic acid), the animals received MS 222 anesthesia and were transcardially perfused. Perfusion and processing of
Correspondence: D.L. Meyer, Zentrum Anatomie, Kreuzbergring 36, 3400 Goettingen, ER.G. 0006-8993/89/$03.50 (~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
390 brains were carried out according to Ebbesson et al. 5. Histological sections reveal that the optic tectum ipsilateral to the injected eye contains anterograde HRP-labeled fibers up to the caudal margin, i.e. in posterior tectal areas not normally receiving a direct uncrossed visual input (Fig. 1). A direct projection to the ipsilateral tectum is restricted to rostral portions of this structure in 3 control specimens of our own (30 days of daily injections of buffer not containing thyroxine), as well as in material of other authors 15. The number of labeled fibers in medial and caudal parts of the ipsilateral tectum is signifi-
Fig. 1. Section through the mesencephalon of Xenopus brain at midtectal level. After thyroxine treatment and injection of HRP into the eye, labeled fibers are present in the lateral optic tract of the ipsilateral optic tectum (arrows). The arrowheads depict fibers which course out of the lateral optic tract. Bar = 100/~m. Low-magnification insert reveals intensive labeling of superficial tectal layers in the tectum contralateral to the injected eye (white arrow).
cantly lower than on the contralateral side, but they are present in all of our specimens treated with thyroxine. The ipsilateral projections can be traced back to the medial and lateral optic tracts. They are not part of a recrossing projection from the other tectum. As depicted in Fig. 2, fibers sprouting from the medial optic tract distribute in central parts of the tectum, whereas, caudal as well as lateral portions of the tectum are targets of fibers emerging from the lateral optic tract. There is no indication for transneuronal transport, such as labeled cells in the tectum or cell bodies containing H R P in structures known to project to the tectum in amphibians 7'1~. In order to test whether the expanded ipsilateral optic tract projection is an immediate effect of thyroxine or whether it depends on the long-term application of this hormone, 3 additional specimens received thyroxine treatment for the 3 days between intraocular H R P injection and time of perfusion. The visual system of these animals resembled that of the controls (see above). Our observations reveal that long-term application of thyroxine can change the connectivity pattern of the CNS in an adult animal. Hitherto, such morphogenetic effects have been known to occur only during certain critical periods during ontogeny in amphibians and other vertebrates, including roam-
Fig. 2. Rostro-lateral view of Xenopus brain. A: arrows depict distribution of uncrossed retinal fibers in the optic tectum of a normal adult frog. The projection is restricted to rostrat portions of the tectum. B: after one month of thyroxine administration, optic tract fibers have sprouted and central as well as caudal parts of the tectum receive axons from the ipsilateral retina.
391 mals. It is well established from experimental evidence and clinical observations that CNS functions can be modified by altered serum levels of thyroxine at any developmental stage, but modifications of connectivity in an adult vertebrate CNS are reported here for the first time. Our data open two lines for further investigation: firstly, it is to be determined, whether some of the known thyroxine effects on functions of the adult mammalian brain may be caused by, as yet undetected, changes in neuronal connectivity. Secondly, the pattern of sprouting in the thyroxine-treated frogs resembles the pattern that exists in a primitive fish 16, suggesting that unnatural hormone availability may re-establish neuronal connectivities lost during the evolutionary development of Xenopus. Ebbesson 3"4 has postulated that induced sprouting does not result in randomly orientated growth and arbitrary new connexions. Rather, he believes that fibers growing under certain artificial circumstances tend to innervate brain structures to which they have a phyiogenetically inherited relationship. Thyroxine may augment affinities which already exist between axons and potential target areas and thus induce the
observed pattern of axonal sprouting. At this point we cannot determine, whether another mechanism may also be involved: there is the possibility that raised serum levels of thyroxine may induce retinal growth. Hence, optic tract fibers terminating in central and caudal portions of the ipsilateral tectum may originate from newly formed ganglion cells. Regardless of the specific details of the mechanisms, our observations have a significant implication with regard to an understanding of the postmetamorphic decrease of serum thyroxine levels in amphibians 6'17. This decrease may not indicate that high thyroxine levels are needed only during metamorphosis 8. Rather, we feel, a frog brain would not be a frog brain, if the adult animal continued to produce significant amounts of this hormone.
1 Allen, B.M., The influence of thyroid gland and hypophysis upon growth and development of amphibian larvea, Endocrinology, 8 (1924) 639-651. 2 DeOlmos, J.S. and Heimer, L., Mapping of collateral projections with the HRP method, Neurosci. Lett., 6 (1977) 107-114. 3 Ebbesson, S.O.E., The parcellation theory and its relation to interspecific variability in brain organization, evolutionary and ontogenetic development and neuronal plasticity, Cell Tissue Res., 213 (1980) 179-212. 4 Ebbesson, S.O.E., Evolution and ontogeny of neural circuits, Behav. Brain Sci., 7 (1984) 321-366. 5 Ebbesson, S.O.E., Hansel, M. and Scheich, H., An 'on the slide' modification of the DeOlmos-Heimer HRP method, Neurosci. Lett., 22 (1981) 1-4. 6 Etkin, W., How a tadpole becomes a frog, Sci. Am., 214 (1966) 76-88. 7 Finkenstaedt, T., Ebbesson, S.O.E. and Ewert, J.-P., Projections to the midbrain tectum in Salamandra salamandra L., Cell Tissue Res., 234 (1983) 39-55. 8 Fleischmann, W., Comparative physiology of the thyroid hormone, Q. Rev. Biol., 22 (1947) 119-140. 9 Giguere, V., Ong, E.S., Segui, P. and Evans, R.M., Identification of a receptor for the morphogen retinoic acid, Nature (Lond.), 330 (1987) 624-629. 10 Hoskins, S.G., Control of the development of the ipsilat-
eral retinothaIamic projection in Xenopus laevis by thyroxine, J. Neurobiol., 17 (1986) 203-229. Hoskins, S.G. and Grobstein, P., Induction of the ipsilateral retinothalamic projection in Xenopus laevis by thyroxine, Nature (Lond.), 307 (1984) 730-733. Hoskins, S.G. and Grobstein, P., Development of the ipsilateral retinothalamic projection in the frog Xenopus laevis. III. The role of thyroxine, J. Neurosci., 5 (1985) 930-940. Jacobson, M., Developmental Neurobiology, Plenum, New York, 1978. LaVail, J.H. and LaVail, M.M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. Levine, R.L., An autoradiographic study of the retinal projections in Xenopus laevis with comparisons to Rana, J. Comp. Neurol., 189 (1980) 1-29. Northcutt, R.G. and Butler, A.B., Retinofugal pathways in the longnose gar, Lepisosteus osseus, J. Comp. Neurol., 166 (1976) 1-16. Rosenkilde, P., Role of feedback in amphibian thyroid regulation, Fortschr. Zool., 22 (1974) 99-116. Wilczynski, W. and Northcutt, R.G., Afferents to the optic rectum of the leopard frog: an HRP study, J. Comp. Neurol., 173 (1977) 219-230.
We thank S. Koetting and C. Sebralla for expert technical assistance and Dr. T.H. Bullock (La Jolla), Dr. W. Breipohl (Brisbaine), Dr. J. Corwin (Charlottesville) and Dr. H. Scheich (Darmstadt) for critically reading the manuscript. Supported by Deutsche Forschungsgemeinschaft grants to A.M. and D.L.M.
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389
BRE 23608
Thyroxine influences neuronal connectivity in the adult frog brain M.H. Hofmann, A. Michler and D.L. Meyer Department of Anatomy, School of Medicine, Universityof Goettingen, Goettingen (E R. G.) (Accepted 28 March 1989) Key words: Thyroxine; Visual system; Optic tectum; Neuronal sprouting; Neuronal plasticity; Amphibian
In contrast to results of earlier investigations the influence of thyroxine on CNS connectivity is not restricted to circum-metamorphic stages in frogs. Neuroanatomical findings in adult Xenopus treated with thyroxine reveal a spread of the ipsilaterai retino-tectal projection. Sprouting fibers establish a tectal innervation pattern similar to the one found in primitive fish. The question arises, whether thyroxine also has morphogenetic effects in the mature CNS of other species. It has been established that thyroxine influences changes in CNS connectivity, particularly in the visual system, during metamorphosis in amphibians 1' 13. Hoskinsl0 and Hoskins and Grobstein 11'12 demonstrated that the ipsilateral retino-thalamic projection develops, if low dosages of thyroxine are applied to one retina of a tadpole. The local administration of thyroxine did not result in serum levels required to induce metamorphosis, or the rostrally directed translocation of orbits and eyes. These elegant experiments prove that thyroxine does not change the anatomy of the skull until an enlarged binocular visual field has been created, which then might serve as a trigger for the development of uncrossed visual projections. Hence, it can be concluded that thyroxine is directly involved in the growth of some retinal axons that terminate in the ipsilateral half of the brain. Although a variety of observations of thyroxine effects on the developing amphibian brain has been reported, detailed studies of possible morphogenetic effects of this hormone in adult animals are lacking or have led to negative results. It still is generally accepted that the significance of thyroxine for CNS connectivity is limited to the critical period of metamorphosis s, whereas in adult specimens its role appears to be limited to the control of metabolism 9. This assumption is partly based on changes of
thyroxine production levels in amphibians during different developmental stages. Thyroid activity is low until shortly before metamorphosis, then increases during metamorphosis, and rapidly drops to approximately premetamorphic levels after the tadpole has become a frog 6'17. This has led to the conclusion that thyroxine loses much of its significance for the organism once metamorphosis has been completed. To investigate possible effects of thyroxine on the internal morphology of an adult vertebrate brain we injected 8 adult Xenopus of both sexes and 30-40 g b. wt. with a daily dose of 50 ~g of this hormone for 30 days. The thyroxine was dissolved in 0.125 ml S6rensen buffer (0.05 M, pH 7.4) and injected into the dorsal lymphatic sac. The specimens were kept at 24 °C in an aquarium providing a seminatural environment. After the 30 days of treatment, we performed a tracer study using conventional horseradish peroxidase (HRP) techniques 2'14 with a 3-day survival time after unilateral intraocular HRP injection. During this 3-day period, daily thyroxine applications were continued. As during previous intraocular HRP injections (5 /~l of a 30% solution of HRP dissolved in phosphate buffer containing 1% DMSO, 1% lysolecithin, and 1% kainic acid), the animals received MS 222 anesthesia and were transcardially perfused. Perfusion and processing of
Correspondence: D.L. Meyer, Zentrum Anatomie, Kreuzbergring 36, 3400 Goettingen, ER.G. 0006-8993/89/$03.50 (~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)
390 brains were carried out according to Ebbesson et al. 5. Histological sections reveal that the optic tectum ipsilateral to the injected eye contains anterograde HRP-labeled fibers up to the caudal margin, i.e. in posterior tectal areas not normally receiving a direct uncrossed visual input (Fig. 1). A direct projection to the ipsilateral tectum is restricted to rostral portions of this structure in 3 control specimens of our own (30 days of daily injections of buffer not containing thyroxine), as well as in material of other authors 15. The number of labeled fibers in medial and caudal parts of the ipsilateral tectum is signifi-
Fig. 1. Section through the mesencephalon of Xenopus brain at midtectal level. After thyroxine treatment and injection of HRP into the eye, labeled fibers are present in the lateral optic tract of the ipsilateral optic tectum (arrows). The arrowheads depict fibers which course out of the lateral optic tract. Bar = 100/~m. Low-magnification insert reveals intensive labeling of superficial tectal layers in the tectum contralateral to the injected eye (white arrow).
cantly lower than on the contralateral side, but they are present in all of our specimens treated with thyroxine. The ipsilateral projections can be traced back to the medial and lateral optic tracts. They are not part of a recrossing projection from the other tectum. As depicted in Fig. 2, fibers sprouting from the medial optic tract distribute in central parts of the tectum, whereas, caudal as well as lateral portions of the tectum are targets of fibers emerging from the lateral optic tract. There is no indication for transneuronal transport, such as labeled cells in the tectum or cell bodies containing H R P in structures known to project to the tectum in amphibians 7'1~. In order to test whether the expanded ipsilateral optic tract projection is an immediate effect of thyroxine or whether it depends on the long-term application of this hormone, 3 additional specimens received thyroxine treatment for the 3 days between intraocular H R P injection and time of perfusion. The visual system of these animals resembled that of the controls (see above). Our observations reveal that long-term application of thyroxine can change the connectivity pattern of the CNS in an adult animal. Hitherto, such morphogenetic effects have been known to occur only during certain critical periods during ontogeny in amphibians and other vertebrates, including roam-
Fig. 2. Rostro-lateral view of Xenopus brain. A: arrows depict distribution of uncrossed retinal fibers in the optic tectum of a normal adult frog. The projection is restricted to rostrat portions of the tectum. B: after one month of thyroxine administration, optic tract fibers have sprouted and central as well as caudal parts of the tectum receive axons from the ipsilateral retina.
391 mals. It is well established from experimental evidence and clinical observations that CNS functions can be modified by altered serum levels of thyroxine at any developmental stage, but modifications of connectivity in an adult vertebrate CNS are reported here for the first time. Our data open two lines for further investigation: firstly, it is to be determined, whether some of the known thyroxine effects on functions of the adult mammalian brain may be caused by, as yet undetected, changes in neuronal connectivity. Secondly, the pattern of sprouting in the thyroxine-treated frogs resembles the pattern that exists in a primitive fish 16, suggesting that unnatural hormone availability may re-establish neuronal connectivities lost during the evolutionary development of Xenopus. Ebbesson 3"4 has postulated that induced sprouting does not result in randomly orientated growth and arbitrary new connexions. Rather, he believes that fibers growing under certain artificial circumstances tend to innervate brain structures to which they have a phyiogenetically inherited relationship. Thyroxine may augment affinities which already exist between axons and potential target areas and thus induce the
observed pattern of axonal sprouting. At this point we cannot determine, whether another mechanism may also be involved: there is the possibility that raised serum levels of thyroxine may induce retinal growth. Hence, optic tract fibers terminating in central and caudal portions of the ipsilateral tectum may originate from newly formed ganglion cells. Regardless of the specific details of the mechanisms, our observations have a significant implication with regard to an understanding of the postmetamorphic decrease of serum thyroxine levels in amphibians 6'17. This decrease may not indicate that high thyroxine levels are needed only during metamorphosis 8. Rather, we feel, a frog brain would not be a frog brain, if the adult animal continued to produce significant amounts of this hormone.
1 Allen, B.M., The influence of thyroid gland and hypophysis upon growth and development of amphibian larvea, Endocrinology, 8 (1924) 639-651. 2 DeOlmos, J.S. and Heimer, L., Mapping of collateral projections with the HRP method, Neurosci. Lett., 6 (1977) 107-114. 3 Ebbesson, S.O.E., The parcellation theory and its relation to interspecific variability in brain organization, evolutionary and ontogenetic development and neuronal plasticity, Cell Tissue Res., 213 (1980) 179-212. 4 Ebbesson, S.O.E., Evolution and ontogeny of neural circuits, Behav. Brain Sci., 7 (1984) 321-366. 5 Ebbesson, S.O.E., Hansel, M. and Scheich, H., An 'on the slide' modification of the DeOlmos-Heimer HRP method, Neurosci. Lett., 22 (1981) 1-4. 6 Etkin, W., How a tadpole becomes a frog, Sci. Am., 214 (1966) 76-88. 7 Finkenstaedt, T., Ebbesson, S.O.E. and Ewert, J.-P., Projections to the midbrain tectum in Salamandra salamandra L., Cell Tissue Res., 234 (1983) 39-55. 8 Fleischmann, W., Comparative physiology of the thyroid hormone, Q. Rev. Biol., 22 (1947) 119-140. 9 Giguere, V., Ong, E.S., Segui, P. and Evans, R.M., Identification of a receptor for the morphogen retinoic acid, Nature (Lond.), 330 (1987) 624-629. 10 Hoskins, S.G., Control of the development of the ipsilat-
eral retinothaIamic projection in Xenopus laevis by thyroxine, J. Neurobiol., 17 (1986) 203-229. Hoskins, S.G. and Grobstein, P., Induction of the ipsilateral retinothalamic projection in Xenopus laevis by thyroxine, Nature (Lond.), 307 (1984) 730-733. Hoskins, S.G. and Grobstein, P., Development of the ipsilateral retinothalamic projection in the frog Xenopus laevis. III. The role of thyroxine, J. Neurosci., 5 (1985) 930-940. Jacobson, M., Developmental Neurobiology, Plenum, New York, 1978. LaVail, J.H. and LaVail, M.M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. Levine, R.L., An autoradiographic study of the retinal projections in Xenopus laevis with comparisons to Rana, J. Comp. Neurol., 189 (1980) 1-29. Northcutt, R.G. and Butler, A.B., Retinofugal pathways in the longnose gar, Lepisosteus osseus, J. Comp. Neurol., 166 (1976) 1-16. Rosenkilde, P., Role of feedback in amphibian thyroid regulation, Fortschr. Zool., 22 (1974) 99-116. Wilczynski, W. and Northcutt, R.G., Afferents to the optic rectum of the leopard frog: an HRP study, J. Comp. Neurol., 173 (1977) 219-230.
We thank S. Koetting and C. Sebralla for expert technical assistance and Dr. T.H. Bullock (La Jolla), Dr. W. Breipohl (Brisbaine), Dr. J. Corwin (Charlottesville) and Dr. H. Scheich (Darmstadt) for critically reading the manuscript. Supported by Deutsche Forschungsgemeinschaft grants to A.M. and D.L.M.
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