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Presence of retinogeniculate fibers is essential for initiating the formation of each interlaminar space in the lateral geniculate nucleus
123
Developmental Brain Research, 20 (1985) 123-126 Elsevier BRD 60063
Short Communications
Presence of retinogeniculate fibers is essential for initiating the formation of each interlaminar space in the lateral geniculate nucleus J. K. BRUNSO-BECHTOLD and V. A. CASAGRANDE
Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC27103 and Vanderbilt School of Medicine, Nashville, TN 37232 (U. S.A.) (Accepted December 4th, 1984)
Key words: lateral geniculate nucleus - - interlaminar spaces - - visual system - - development
We demonstrated in a previous study that, following neonatal bilateral enucleation in the tree shrew, interlaminar spaces (ILSs) in the dorsal lateral geniculate nucleus fail to form. In the present study we sought to determine which aspects of ILS formation are dependent upon retinal input. Accordingly, we studied the degree of ILS formation in tree shrews which were bilaterally enucleated either during ILS formation on postnatal day 3 (P3) or just after all ILSs were apparent but before they had reached a mature width (P15). Our results indicate that retinal input is necessary for the initial formation of each ILS, but that it is not required for the maturation or maintenance of ILSs which have already begun to form. The lateral geniculate nucleus ( L G N ) in m a n y species (e.g., cat, bushbaby, squirrel, tree shrew, brushtailed possum and n u m e r o u s species of monkeys) is composed of distinct cell layers. In the tree shrew, ferret, squirrel and brushtailed possum these cell layers have been r e p o r t e d to form postnatally2,3,5,7. W e have shown previously that in the tree shrew the formation of the spaces b e t w e e n these cell layers is dep e n d e n t on the presence of retinogeniculate fibers1; this observation has been confirmed in the ferret a. In both experiments, interlaminar spaces (ILSs) failed to form if the eyes were r e m o v e d at birth. If we are to begin to u n d e r s t a n d the mechanism(s) by which retinogeniculate fibers regulate the segregation of L G N cells into layers, we now n e e d to know what aspects of ILS f o r m a t i o n are d e p e n d e n t on the presence of those fibers. In the present study, we sought to d e t e r m i n e whether the presence of retino-
geniculate fibers is essential for the entire t e m p o r a l sequence of ILS f o r m a t i o n or whether, after some critical period in d e v e l o p m e n t , the presence of those fibers is no longer essential for the final m a t u r a t i o n of the ILSs. The normal adult tree shrew L G N has 6 clearly defined cell layers, n u m b e r e d from medial to lateral, which are s e p a r a t e d by relatively cell-free ILSs (Fig. 1). Of these, layers 1 and 5 are innervated by the ipsilateral retina and layers 2, 3, 4, and 6 are innervated by the contralateral retina. These cell layers are not present at birth in the tree shrew and the ILSs between t h e m begin to a p p e a r in the first postnatal week. The ILSs between layers innervated by opposite eyes (e.g., b e t w e e n layers 1 and 2, 4 and 5, and 5 and 6) are the first to d e v e l o p and can be seen by a p p r o x i m a t e l y postnatal day 2 (P2)*, whereas those between layers innervated by the same eye
* We consider the first day of life as postnatal day 0 or P0, the second day as P1, and so on.
Correspondence: J. K. Brunso-Bechtold, Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103, U.S.A. 0165-3806/85/$03.30 (~) 1985 Elsevier Science Publishers B .V. (Biomedical Division)
124
Fig. 1. Horizontal section through the left LGN of a normal adult tree shrew demonstrating the 6 component layers which develop postnatally. Each layer is separated from the adiacent layers by ILSs which develop postnatally. Lateral is at the top and rostra1 LSat the right. Magnification bar,*O.l mm.
(e.g., between layers 2 and 3, and 3 and 4) can first be seen a few days laterz. There are several possible ways in which the retinogeniculate fibers could influence ILS formation.
two infant tree shrews, one on P3 just after the early forming ILSs have appeared but before the late forming ILSs have developed and one on Pi5 after all
They could, for example, be essential only for the initiation of the early forming ILSs after which the rest of the ILSs would form normally in their absence. Or they could be essential for the initiation of each individual ILS after which the final maturation either in
ILSs have become apparent but before they have reached their mature width. An unoperated littermate of each of these animals was perfused at the time of enucleation to enable us to determine the exact state of ILS formation at which the operated animals were enucleated. The operated animals were
length (parallel to the layer) or width of the ILSs would take place normally in their absence. The retinogeniculate fibers could also be essential for the entire maturation of the ILSs in which case no ILS development or further maturation would take place after their removal. Finally, they could be essential for the continued maintenance of the ILSs such that ‘complete removal of the retinogeniculate fibers in the infant animal would result in a deterioration of the ILSs which had already formed. To address these issues, we bilaterally enucleated
anesthetized with chloral hydrate, bilaterally enucleated1 and allowed to survive to sexual maturity (3 months). At maturity, the animals were anesthetized with sodium pentobarbital and killed by transcardial perfusion of saline followed by 10% formol-saline. The brains of both experimental and control animals were embedded in celloidin, horizontally sectioned at 30 pm and stained with thionin. Fig. 2A shows the degree of LGN laminar development in the normal animal perfused on P3. At this stage, the early forming ILSs indicated by the arrows
125 are the only ones that clearly are present. A compa-
formation of each ILS. This observation is consistent
rable section through the L G N of the adult that was
with observations in the rhesus m o n k e y in which the
bilaterally enucleated on P3 when its littermate was
degree of L G N lamination in the adult has been re-
perfused is shown in Fig. 2B. As in the L G N of the
ported to be similar to what was presumed to be pres-
infant per'fused on P3, there are two ILSs (arrows)
ent at the time of unilateral enucleation6. In the pres-
present in the same relative position in the nucleus.
ent study, by using bilateral enucleation, we ensure
In the L G N of the bilateral enucleate, however, the ILSs are considerably wider and the cells appear more mature than in the normal animal on P3. Fig. 2C shows the L G N from the normal animal perfused on P15. All layers are apparent at this stage although the ILSs are quite narrow and i m m a t u r e in appearance. For reference, the arrows indicate the same laminar borders as in Fig. 2A, B. A comparable section through the L G N of the adult that was bilaterally enucleated on P15 when its littermate was perfused is shown in Fig. 2D. As in the L G N of the animal perfused on P15, all of the ILSs in this section can be distinguished. In the L G N of the bilateral enucleate, however, the ILSs are of normal width and the cells appear to be mature (cf. Fig. 1). These results suggest that the presence of retinogeniculate fibers is essential for the initiation of the
Fig. 2. A: horizontal section through the LGN from a normal tree shrew sacrificed on P3 when only the early forming ILSs are apparent. The ILS developing between presumptive layers 1 and 2 is indicated by the lower arrow and that between presumptive layers 4 and 5 is indicated by the upper arrow; the decreased density just below the upper arrow is the beginning of the formation of a bilayer in layer 4 (cf. Fig. 1), B: horizontal section through the LGN of an adult tree shrew which was bilaterally enucleated at the same time (P3) as its littermate, shown in 2A, was sacrificed. The only clear laminar borders (indicated by the arrows) are those which had begun to form at the time of enucleation. C: horizontal section through the LGN of a tree shrew sacrificed on P15, after all ILSs can be distinguished. The lower and upper arrows again indicate the borders between layers 1 and 2 and layers 4 and 5, respectively. D: horizontal section through the LGN of an adult tree shrew which was bilaterally enucleated at the same time (P15) as its littermate, shown in 2C, was sacrificed. The arrows indicate the same borders as those in 2C. Note that, as in the normal adult, all 6 layers can be clearly distinguished. In each plate, lateral is at the top and rostral is at the right; magnification bar in each plate, 0.1 mm.
126 that the ILSs which were present at the time of enucteation are not maintained by the fibers from the remaining eye. Thus, our findings indicate that the ~ransient presence of retinogenicutate fibers during the development of the early forming ILSs is insufficient for complete ILS formation. Instead, the formation of each gap that initially defines a developing ILS requires the presence of the retinogeniculate fibers and, accordingly, no ILS will form if the gap is not already present at the time retinogeniculate fibers are removed. The presence of retinogeniculate fibers is not necessary, however, for the final widening and subsequent maintenance of the ILSs. Consequently, once individual ILSs have begun to develop they continue to widen even if the retinogeniculate fibers are removed. The significance of these observations is that we now know which phases of ILS formation are
1 Brunso-Bechtold, J. K. and Casagrande, V. A., Effect of bilateral enucleation on the development of layers in the dorsal lateral geniculate nucleus, Neuroscience, 6 (1981) 2579-2586. 2 Brunso-Bechtold, J. K. and Casagrande, V. A., Early postnatal development of laminar characteristics in the dorsal lateral geniculate nucleus of the tree shrew, J. Neurosci., 2 (1982) 589-597. 3 Cusick, C. G. and Kaas, J. H., Retinal projections in adult and newborn grey squirrels, Develop. Brain Res., 4 (1981) 275-284. 4 LaMantia, A.-S. and Guillery, R. W., The effects of binocular enucleation on the development of the dorsal lateral ge-
dependent on retinogeniculate input. For example, the growth promoting or 'trophic' influence that the retinogeniculate fibers may have is not essential for the maturation or widening of the newly formed, narrow gaps between adjacent L G N layers. Nevertheless, we now know that the retinogeniculate fibers play a critical role in the developmental mechanism(s) that regulate the initial segregation of L G N cells into individual layers. The authors wish to thank Dr. Ed Debruyn and Keith Sutton for editorial comments, Elizabeth Birecree, D o n n a Moore Smith and Constance Linville for technical assistance, and D a p h n e Styers for careful typing of the manuscript. This work was supported by N I H Grants EY0 5028, EY0 3881, EY0 5038 and K04-EY0 0223.
niculate nucleus (E)LGN) of the ferret, Soc. Neurosci. Abstr., 8 (1982) 814.
5 Linden, D. C., Guillery, R. W. and Cucchiaro, J., The dorsal lateral geniculate nucleus of the normal ferret and its postnatal development, J. comp. Neurol., 203 (1981) 189-211. 6 Rakic, P., Development of visual centers in the primate brain depends on binocular competition before birth, Science, 214 (1981) 928-930. 7 Sanderson, K. J., Dixon, P. G. and Pearson, L. J., Postnatal development of retinal projections in brushtailed possum, Trichosurus vulpecula, Develop. Brain Res., 5 (1982) 161-180.
Developmental Brain Research, 20 (1985) 123-126 Elsevier BRD 60063
Short Communications
Presence of retinogeniculate fibers is essential for initiating the formation of each interlaminar space in the lateral geniculate nucleus J. K. BRUNSO-BECHTOLD and V. A. CASAGRANDE
Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC27103 and Vanderbilt School of Medicine, Nashville, TN 37232 (U. S.A.) (Accepted December 4th, 1984)
Key words: lateral geniculate nucleus - - interlaminar spaces - - visual system - - development
We demonstrated in a previous study that, following neonatal bilateral enucleation in the tree shrew, interlaminar spaces (ILSs) in the dorsal lateral geniculate nucleus fail to form. In the present study we sought to determine which aspects of ILS formation are dependent upon retinal input. Accordingly, we studied the degree of ILS formation in tree shrews which were bilaterally enucleated either during ILS formation on postnatal day 3 (P3) or just after all ILSs were apparent but before they had reached a mature width (P15). Our results indicate that retinal input is necessary for the initial formation of each ILS, but that it is not required for the maturation or maintenance of ILSs which have already begun to form. The lateral geniculate nucleus ( L G N ) in m a n y species (e.g., cat, bushbaby, squirrel, tree shrew, brushtailed possum and n u m e r o u s species of monkeys) is composed of distinct cell layers. In the tree shrew, ferret, squirrel and brushtailed possum these cell layers have been r e p o r t e d to form postnatally2,3,5,7. W e have shown previously that in the tree shrew the formation of the spaces b e t w e e n these cell layers is dep e n d e n t on the presence of retinogeniculate fibers1; this observation has been confirmed in the ferret a. In both experiments, interlaminar spaces (ILSs) failed to form if the eyes were r e m o v e d at birth. If we are to begin to u n d e r s t a n d the mechanism(s) by which retinogeniculate fibers regulate the segregation of L G N cells into layers, we now n e e d to know what aspects of ILS f o r m a t i o n are d e p e n d e n t on the presence of those fibers. In the present study, we sought to d e t e r m i n e whether the presence of retino-
geniculate fibers is essential for the entire t e m p o r a l sequence of ILS f o r m a t i o n or whether, after some critical period in d e v e l o p m e n t , the presence of those fibers is no longer essential for the final m a t u r a t i o n of the ILSs. The normal adult tree shrew L G N has 6 clearly defined cell layers, n u m b e r e d from medial to lateral, which are s e p a r a t e d by relatively cell-free ILSs (Fig. 1). Of these, layers 1 and 5 are innervated by the ipsilateral retina and layers 2, 3, 4, and 6 are innervated by the contralateral retina. These cell layers are not present at birth in the tree shrew and the ILSs between t h e m begin to a p p e a r in the first postnatal week. The ILSs between layers innervated by opposite eyes (e.g., b e t w e e n layers 1 and 2, 4 and 5, and 5 and 6) are the first to d e v e l o p and can be seen by a p p r o x i m a t e l y postnatal day 2 (P2)*, whereas those between layers innervated by the same eye
* We consider the first day of life as postnatal day 0 or P0, the second day as P1, and so on.
Correspondence: J. K. Brunso-Bechtold, Department of Anatomy, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103, U.S.A. 0165-3806/85/$03.30 (~) 1985 Elsevier Science Publishers B .V. (Biomedical Division)
124
Fig. 1. Horizontal section through the left LGN of a normal adult tree shrew demonstrating the 6 component layers which develop postnatally. Each layer is separated from the adiacent layers by ILSs which develop postnatally. Lateral is at the top and rostra1 LSat the right. Magnification bar,*O.l mm.
(e.g., between layers 2 and 3, and 3 and 4) can first be seen a few days laterz. There are several possible ways in which the retinogeniculate fibers could influence ILS formation.
two infant tree shrews, one on P3 just after the early forming ILSs have appeared but before the late forming ILSs have developed and one on Pi5 after all
They could, for example, be essential only for the initiation of the early forming ILSs after which the rest of the ILSs would form normally in their absence. Or they could be essential for the initiation of each individual ILS after which the final maturation either in
ILSs have become apparent but before they have reached their mature width. An unoperated littermate of each of these animals was perfused at the time of enucleation to enable us to determine the exact state of ILS formation at which the operated animals were enucleated. The operated animals were
length (parallel to the layer) or width of the ILSs would take place normally in their absence. The retinogeniculate fibers could also be essential for the entire maturation of the ILSs in which case no ILS development or further maturation would take place after their removal. Finally, they could be essential for the continued maintenance of the ILSs such that ‘complete removal of the retinogeniculate fibers in the infant animal would result in a deterioration of the ILSs which had already formed. To address these issues, we bilaterally enucleated
anesthetized with chloral hydrate, bilaterally enucleated1 and allowed to survive to sexual maturity (3 months). At maturity, the animals were anesthetized with sodium pentobarbital and killed by transcardial perfusion of saline followed by 10% formol-saline. The brains of both experimental and control animals were embedded in celloidin, horizontally sectioned at 30 pm and stained with thionin. Fig. 2A shows the degree of LGN laminar development in the normal animal perfused on P3. At this stage, the early forming ILSs indicated by the arrows
125 are the only ones that clearly are present. A compa-
formation of each ILS. This observation is consistent
rable section through the L G N of the adult that was
with observations in the rhesus m o n k e y in which the
bilaterally enucleated on P3 when its littermate was
degree of L G N lamination in the adult has been re-
perfused is shown in Fig. 2B. As in the L G N of the
ported to be similar to what was presumed to be pres-
infant per'fused on P3, there are two ILSs (arrows)
ent at the time of unilateral enucleation6. In the pres-
present in the same relative position in the nucleus.
ent study, by using bilateral enucleation, we ensure
In the L G N of the bilateral enucleate, however, the ILSs are considerably wider and the cells appear more mature than in the normal animal on P3. Fig. 2C shows the L G N from the normal animal perfused on P15. All layers are apparent at this stage although the ILSs are quite narrow and i m m a t u r e in appearance. For reference, the arrows indicate the same laminar borders as in Fig. 2A, B. A comparable section through the L G N of the adult that was bilaterally enucleated on P15 when its littermate was perfused is shown in Fig. 2D. As in the L G N of the animal perfused on P15, all of the ILSs in this section can be distinguished. In the L G N of the bilateral enucleate, however, the ILSs are of normal width and the cells appear to be mature (cf. Fig. 1). These results suggest that the presence of retinogeniculate fibers is essential for the initiation of the
Fig. 2. A: horizontal section through the LGN from a normal tree shrew sacrificed on P3 when only the early forming ILSs are apparent. The ILS developing between presumptive layers 1 and 2 is indicated by the lower arrow and that between presumptive layers 4 and 5 is indicated by the upper arrow; the decreased density just below the upper arrow is the beginning of the formation of a bilayer in layer 4 (cf. Fig. 1), B: horizontal section through the LGN of an adult tree shrew which was bilaterally enucleated at the same time (P3) as its littermate, shown in 2A, was sacrificed. The only clear laminar borders (indicated by the arrows) are those which had begun to form at the time of enucleation. C: horizontal section through the LGN of a tree shrew sacrificed on P15, after all ILSs can be distinguished. The lower and upper arrows again indicate the borders between layers 1 and 2 and layers 4 and 5, respectively. D: horizontal section through the LGN of an adult tree shrew which was bilaterally enucleated at the same time (P15) as its littermate, shown in 2C, was sacrificed. The arrows indicate the same borders as those in 2C. Note that, as in the normal adult, all 6 layers can be clearly distinguished. In each plate, lateral is at the top and rostral is at the right; magnification bar in each plate, 0.1 mm.
126 that the ILSs which were present at the time of enucteation are not maintained by the fibers from the remaining eye. Thus, our findings indicate that the ~ransient presence of retinogenicutate fibers during the development of the early forming ILSs is insufficient for complete ILS formation. Instead, the formation of each gap that initially defines a developing ILS requires the presence of the retinogeniculate fibers and, accordingly, no ILS will form if the gap is not already present at the time retinogeniculate fibers are removed. The presence of retinogeniculate fibers is not necessary, however, for the final widening and subsequent maintenance of the ILSs. Consequently, once individual ILSs have begun to develop they continue to widen even if the retinogeniculate fibers are removed. The significance of these observations is that we now know which phases of ILS formation are
1 Brunso-Bechtold, J. K. and Casagrande, V. A., Effect of bilateral enucleation on the development of layers in the dorsal lateral geniculate nucleus, Neuroscience, 6 (1981) 2579-2586. 2 Brunso-Bechtold, J. K. and Casagrande, V. A., Early postnatal development of laminar characteristics in the dorsal lateral geniculate nucleus of the tree shrew, J. Neurosci., 2 (1982) 589-597. 3 Cusick, C. G. and Kaas, J. H., Retinal projections in adult and newborn grey squirrels, Develop. Brain Res., 4 (1981) 275-284. 4 LaMantia, A.-S. and Guillery, R. W., The effects of binocular enucleation on the development of the dorsal lateral ge-
dependent on retinogeniculate input. For example, the growth promoting or 'trophic' influence that the retinogeniculate fibers may have is not essential for the maturation or widening of the newly formed, narrow gaps between adjacent L G N layers. Nevertheless, we now know that the retinogeniculate fibers play a critical role in the developmental mechanism(s) that regulate the initial segregation of L G N cells into individual layers. The authors wish to thank Dr. Ed Debruyn and Keith Sutton for editorial comments, Elizabeth Birecree, D o n n a Moore Smith and Constance Linville for technical assistance, and D a p h n e Styers for careful typing of the manuscript. This work was supported by N I H Grants EY0 5028, EY0 3881, EY0 5038 and K04-EY0 0223.
niculate nucleus (E)LGN) of the ferret, Soc. Neurosci. Abstr., 8 (1982) 814.
5 Linden, D. C., Guillery, R. W. and Cucchiaro, J., The dorsal lateral geniculate nucleus of the normal ferret and its postnatal development, J. comp. Neurol., 203 (1981) 189-211. 6 Rakic, P., Development of visual centers in the primate brain depends on binocular competition before birth, Science, 214 (1981) 928-930. 7 Sanderson, K. J., Dixon, P. G. and Pearson, L. J., Postnatal development of retinal projections in brushtailed possum, Trichosurus vulpecula, Develop. Brain Res., 5 (1982) 161-180.