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Prenatal Development and Reorganization in the Visual System of the Cat

L. M. Chalupa and R. W. Williams
Department of Psychology, University of California, Davis, California 95616, USA
 

Development of Visual Pathways in Mammals, pages 89–102
Stone J., Dreher G., and Rapaport D., editors
© 1984 Alan R. Liss, New York


 

LM Chalupa

 

Note to the reader: This review summarizes the first phase of work by Leo Chalupa and colleagues on the early development of cat retinal projections. If you select a figure, a higher-quality image will download into a separate window. Move this window to the side.


 


Four years ago we initiated a research program which sought to answer two questions:

 

  1. What are the events in fetal life which give rise to mature patterns of connection in the mammalian visual system?
  2. To what degree is the maturation of the visual system perturbed by interrupting binocular interactions at an early stage of development?

 

Before summarizing the progress we have made in answering these questions, it is worth considering why we chose to study cats. First, since the introduction of the monocular deprivation paradigm by Wiesel and Hubel (1963), much has been learned about normal and abnormal postnatal development of vision in cats, and this information provides a foundation for investigating the early development of this species’ visual system. Second, the cat is multiparous, and it is therefore possible to acquire a sufficient number of fetuses of known gestational age. Third, the 63–65 day gestation of the cat is long enough to allow adequate temporal resolution of various events that occur during prenatal development. Fourth, the size of the fetal cat’s brain is comparatively large, and thus accessible to various types of manipulations.

We began our studies by tracing the development of retinal projections to the main visual centers, using the anterograde transport of horseradish peroxidase and tritiated leucine. One of the tracers was injected into the right eye, and the other tracer into the left eye, of fetuses of known gestational age. Pregnancy was timed by exposing an estrous female to a potent male for 24 hours. Several matings were observed during this period, and the end of the 24 hour exposure was considered to mark the first day of gestation (embryonic day 1, or El).

Figure 1 Figure 1. Retinogeniculate projections at E38 and E46 in fetal cats demonstrated by the anterograde transport of HRP. Coronal sections; the contralateral retinal input is shown to the left; ipsilateral is to the right. In contrast to Shatz (1983), we find almost total overlap of crossed and uncrossed retinal projections at E46 (see Fig. 2, especially).

Our earliest successful injections were made on E38 (Fig. 1). At this age virtually all ganglion cells have been generated (Walsh et al. 1983), ganglion cell growth cones have extended through the optic nerve (Williams et al. 1983a), and at least some of the axons have reached as far as the posterior margin of the superior colliculus (Williams, Chalupa 1982a). As shown in figure 1, as early as E38 there is substantial overlap of projections from right and left eyes. The contralateral fiber influx, however, is much heavier (Fig. 1A) than the ipsilateral (Fig. lB). Furthermore, it appears that only a fraction of the retinal ingrowth has yet entered the terminal fields by this age.

Between E38 and E56 there is a gradual elaboration and restructuring of retinal projections. The density of terminal label becomes much greater, and by E46 virtually the entire lateral geniculate nucleus (Fig. 2), pretectum (Williams, Chalupa 1983a), and superior colliculus (Williams, Chalupa 1982a) receive heavy projections from both eyes. Overlap of projections from right and left eyes is 100% in the geniculate nucleus (Fig. 2). Virtually complete overlap has also been described in the lateral geniculate of another carnivore, the ferret (Linden et al. 1981), and in the monkey (Rakic 1977). (Our findings in the cat differ, however, from those of Shatz (1983), who reported that at all fetal ages, including E46, most of the cat’s geniculate nucleus is innervated only by fibers from the contralateral eye.) It is crucial to note that at E46 the pattern of labeling is not uniform. In the geniculate nucleus the densities of the crossed and uncrossed retinal projections appear to vary inversely. This pattern foreshadows the development of discrete layers that contain terminals of either the right or the left eye. As shown in figure 3, by E56 segregation is essentially complete.

Figure 2 Figure 2. Fig. 2. Overlap of projections from right and left eyes to the lateral geniculate nucleus at E46. The contralateral eye received an injection of 3H-leucine; the ipsilateral eye received an injection of HRP. Therefore, panels A, C, and E show the crossed input and the other three panels (B, D, and F) show the uncrossed input. Pairs of adjacent sections are shown under darkfield illumination at three representative rostrocaudal levels: A and B are at the caudal pole; C and D are central; E and F are rostral. Shrinkage during processing was greater in HRP sections than of autoradiographic sections. Therefore, the magnification of each pair of micrographs was adjusted so as to obtain the most accurate match between tissue landmarks. Although overlap is virtually 100%, note that the density of labeling is not uniform. Segregation is incipient at this age. A, B, H and F at X40–60; C and D at X16–20.
 

There are significant differences in the gestational age at which segregation is achieved in different nuclei. Segregation starts and ends more than a week earlier in the geniculate nucleus than in either the superior colliculus or the pretectum (see Williams and Chalupa, 1983a). Furthermore, the separation of retinal fibers takes place later in the C laminae than in the A laminae of the geniculate, as was originally suggested by Shatz (1983). Since only the A laminae receive a dominant input from the beta class of ganglion cells, these differences may be related to the differential maturation of the major ganglion cell types. Medium-sized retinal ganglion cells–presumably the beta class, that project heavily to the dorsal layers of the adult geniculate–are mostly generated before the small cells of the gamma class, that project to the superior colliculus, pretectum and C layers of the geniculate (Polley et al. 1981; Kliot, Shatz 1982; Walsh et al. 1983).

Figure 3

Fig. 3. Segregation of the crossed and uncrossed retinogeniculate influx at E56. Darkfield micrographs of peroxidase reaction product. A. Crossed projection; B. Uncrossed projection.


 

During gestation there is a marked overproduction of ganglion cell axons (Ng, Stone 1982; Williams, 1983; Williams et al., 1983a,b; R.W. Williams, M.J. Bastiani, and L.M. Chalupa, in progress). Using a quantitative electron microscopic method, we have found that the first 100 ganglion cell axons venture into the optic stalk on E19. Four days later, about 1,000 fibers are present. By E28 the number of axons has increased to approximately 40,000. At E28 and E33 the counts include a substantial number of growth cones (Fig. 4). For instance, at E33, 3% (n = 8,000) of the neurites are growth cones. These are characterized by extensive veil-like processes, some of which extend radially four microns from the core (Fig. 4). Growth cones tend to grow around the perimeter of the nerve, in apposition to glial processes. However, some extending axons are also found in the central region, without apparent connection to glial precursors.

The peak axon count of 660,000 is attained at E39. At this age only a few hundred growth cones were noted. A population of more than 500,000 is maintained until E44. Between E44 and E48 there is a precipitous loss of fibers. By E48 the axon complement has eroded to 330,000. Thereafter the loss of axons is more gradual; the adult population of about 160,000 is not reached until at least several weeks after birth.

The excess axons produced early in development could potentially contribute to the wide distribution of retinal projections observed during fetal life. However, the initial massive loss of axons, which begins even before E44, precedes by several days the onset of segregation in the geniculate nucleus. Moreover, fiber loss is still occurring during the first postnatal month, long after the segregation of all retinal efferents is complete. Thus, while fiber loss may underlie the segregation of early retinal projections, we suspect that there are other features of the developing visual system that are shaped by this phenomenon.

 

Early Termination of Binocular Competition

 

Our second objective was to determine the contributions made by prenatal binocular competition to the development of the cat’s visual pathways. For this purpose we removed one eye from fetuses at known gestational ages (between E40 and E56), and when these animals reached maturity we studied the organization of their visual systems.

Figure 8

Fig. 4. Growth cones and axons in a peripheral fasciculus of the optic nerve at E33. To the right, dark astroblastic processes separate the fascicles and form a limiting membrane around the nerve, the edge of which crosses the lower left corner. Some of the very large growth cones are marked with asterisks. Calibration bar is 1 µm. Download a high-resolution 300 KB image.

In these enucleated animals, the remaining retina innervates the entire ipsilateral and contralateral geniculate nuclei (Fig. 5) (Williams, Chalupa 1982b & 1983b; Williams 1983). However, as shown in figure 5B, the geniculate ipsilateral to the remaining eye is substantially smaller than that on the contralateral side. The morphology of the lateral geniculate nuclei of these one-eyed cats is characterized by two laminae: a dorsal magnocellular layer and a ventral layer. A similar result has been described by Rakic (1981) in prenatally enucleated monkeys. Presumably, the magnocellular layer corresponds to what would normally have been layers A and Al, whereas the ventral layer corresponds to what would have been subdivided into layers C, C1, C2, and C3. Thus, the six laminae of the normal cat’s geniculate are supplanted by two composite layers.

We have also shown that there are significantly more optic nerve fibers in prenatally enucleated cats than is normal (Williams et al. 1983b). Furthermore, the number of fibers within the optic nerve of these animals matches the number of ganglion cells in the remaining retina (Henderson et al. 1983; Chalupa et al., in review). The results of these experiments, summarized in table 1, show that the prenatally enucleated cats have about 20% or 30,000 more ganglion cells than normal animals. Obviously this excess could contribute to the widespread retinal projections found in one-eyed cats.

An unexpected result of this study was that the number of ganglion cells saved does not depend critically upon the gestational age at which enucleations are performed. Removal of an eye at E42, when there are more than 500,000 fibers in the optic nerve, is no more effective in saving ganglion cells from imminent death than is enucleation at E51, when there are 300,000 axons.

Figure 5

Fig. 5. Retinogeniculate projections in an adult cat from which one eye was removed on E49. The crossed projection is shown in A; the uncrossed in B. parts of both nuclei are labeled with the peroxidase chromogen, however, the ipsilateral nucleus and the ipsilateral retinal projection are considerably smaller.


 

 


TABLE 1: Effects of Prenatal Unilateral Enucleation upon Ganglion Cell and Optic Axon Number


  Ganglion Cell     Axon
Animal* Number     Number

Control 1 151,000 (R)**     —-
  152,000 (L)     —-
Control 2 —-     158,000
Control 3 —-     159,000
Control 4       164,000

E42 Enucleate 180,000     178,000
E51 Enucleate 182,000     190,000
E45 Enucleate —-     198,000
E46 Enucleate —-     200,000

*All animals were mature
**(R) right retina; (L) left retina

 


 

Early eye removal has been shown to maintain widespread retinal projections in a number of mammalian species (e.g., Chow et al. 1973; Sanderson 1978; Land, Lund 1979; Frost, Schneider 1979 Rakic 1981); however, relatively little is know about the functional organization of the altered retinal connections (but see Rhoades, Chalupa 1980). Accordingly, we sought to determine the functional properties of the retinal influx to the geniculate of prenatally enucleated cats. For this purpose, extracellular single cell recordings were made within the lateral geniculate, contralateral and ipsilateral to the remaining eye of adult animals from which an eye had been removed more than two weeks before birth. The results are clear-cut: all regions of the geniculate nuclei of these cats are functionally innervated by the retina input, and the topographic organization is similar to that of the normal cat (Williams, Chalupa 1982 & 1983b; Williams 1983). Furthermore, injection of peroxidase into the medial region of the geniculate that contains the area centralis representation revealed a normal decussation pattern in the remaining retina (Williams, Chalupa 1983b). This result rules out the possibility that an aberrant retinal input from the inappropriate hemiretina was either functionally suppressed or ineffective in driving thalamic units. Parenthetically, it should be noted, that we have found recently a clear decussation pattern following unilateral injection of HRP as early as E44 (Lia et al., 1983).

Even though the functional organization of the lateral geniculate nucleus appeared normal in prenatally enucleated cats, it seemed important to also examine the properties of the visual cortex in these animals. Studies of the visual cortex were carried out in collaboration with Drs. Brenda Shook and Lamberto Maffei (B.L. Shook, L. Maffei, and L.M. Chalupa, 1983). Small iontophoretic deposits of horseradish peroxidase conjugated to wheat germ agglutinin were made into the A lamina of normal cats and into the most dorsal portion of the geniculate nuclei of enucleated animals. As expected, discrete patches of label were found in layer IV of normal cats, whereas continuous label was found in the enucleated cats. Thus, in agreement with the previous work of Rakic (1981), ocular dominance columns fail to develop following early removal of one eye.

In the same cats long tangential microelectrode penetrations were made through area 17. Most of these penetrations extended 2.5–3.0 mm at an oblique angle down the medial bank of the marginal gyrus, and thus the electrode traversed a region that in normal cats is occupied by a number of discrete ocular dominance columns. In the prenatally enucleated cats, the remaining eye could activate all neurons, and the activity did not show any signs of waxing and waning. In contrast, in animals enucleated as adults, regions of reduced visual activity were recorded when similar penetrations were made. It is particularly noteworthy that cortical neurons of prenatally enucleated animals exhibited an orderly progression in orientation selectivity. In several penetrations, sequences of cells were encountered that had a 180 degree cycle of preferred orientations. This is characteristic of hypercolumns in the visual cortex of normal animals (Hubel, Wiesel 1962). Therefore, this finding indicates that orientation columns can develop independently of ocular dominance columns.

There was one significant difference in the visual receptive field properties between prenatally enucleated and normal cats. Within 50 of the area centralis representation–where most penetrations were confined–the dimensions of receptive fields were significantly smaller in prenatally enucleated animals than in normal cats. One possible explanation for this intriguing finding is that dendritic fields of ganglion cell may be smaller than normal. We know that the remaining retina of these animals has about 30,000 more ganglion cells than normal, yet the retinal area is not appreciably larger. One would anticipate that exacerbated dendro-dendritic competition among ganglion cells in the remaining retina (see Perry, Linden 1982) would result in a reduction in the size of dendritic fields. Such a reduction would permit accommodation of the excess ganglion cell complement without disrupting the retinal mosaic.

 

ACKNOWLEDGEMENT: Supported by grants EY03391 and EY05670 from the National Eye Institute of NIH.

 

REFERENCES

Chalupa LM, Williams RW, Henderson Z. Binocular competition in the fetal cat regulates the size of the ganglion cell population. Neuroscience 12:1139–1146.

Chow EL, Mathers LH, Spear PD (1973) Spreading of uncrossed retinal projection in superior colliculus of neonatally enucleated rabbits. J Comp Neurol 150:307.

Frost DO, Schneider GE (1979) Plasticity of retinofugal projections after partial lesions of the retina in newborn Syrian hamsters. J Comp Neurol 185:517

Henderson Z, Williams RW, Chalupa LM (1983) Removal on an eye in the fetal cat increases the number of ganglion cells maintained in the remaining retina. Invest Ophthalmol Vis Sci 24:290.

Hubel DH, Wiesel TN (1962) Receptive field, binocular interaction, and functional architecture in the cat’s visual cortex. J Physiol (Lond) 160:106.

Kliot M, Shatz CJ (1982) Genesis of different retinal ganglion cell types in the cat. Soc Neurosci Abst 8:815.

Land PW, Lund RD (1979) Development of the rat’s uncrossed retinotectal pathway and its relation to plasticity studies. Science 205:698.

Lia B, Williams RW, Chalupa LM (1983). Early development of retinal specialization: the distribution and decussation patterns of ganglion cells in the prenatal cat demonstrated by retrograde peroxidase labelling. Soc Neurosci Abstr 9:702.

Linden DC, Guillery RW, Cucchiaro J (1981) The dorsal lateral geniculate nucleus of the normal ferret and its postnatal development. J Comp Neurol 203:189.

Ng AYK, Stone J (1982) The optic nerve of the cat: Appearance and loss of axons during normal development. Develop Brain Res 5:263.

Perry VH, Linden R (1982) Evidence for dendritic competition in the developing retina. Nature 297:683.

Polley EH, Walsh C, Hickey TL (1981) Neurogenesis in cat retina: A study using 3H-thymidine autoradiography. Soc Neurosci Abstr 7:672.

Rakic P (1977) Prenatal development of the visual system in rhesus monkey. Phil Trans R Soc Lond B 278:245.

Rakic P (1981) Development of visual centers in the primate brain depends on binocular competition before birth. Science 214:928.

Rhoades RW, Chalupa LM (1980) Effects of neonatal enucleation on receptive-field properties of visual neurons in superior colliculus of the golden hamster. J Neurophysiol 43:595.

Sanderson KJ, Pearson LJ, Dixon PG (1978) Altered retinal projections in brushtailed possum, Trichosurus vupecula, following removal of one eye. J Comp Neurol 143:101.

Shatz CJ (1983) The prenatal development of the cat’s retinogeniculate pathway. J Neurosci 3:482.

Shook BL, Maffei L, Chalupa LM (1983) Functional organization of the cat’s visual cortex after prenatal unilateral enucleation. Soc Neurosci Abst 9: in press.

Walsh C, Polley EH, Hickey TL, Guillery RW (1983) Generation of cat retinal ganglion cells in relation to central pathways. Nature 302:611.

Wiesel TN, Hubel DH (1963) Single-cell responses in striate cortex of kittens deprived of vision in one eye. J Neurophysiol 26:1003.

Williams RW, Chalupa LM (1982a) Prenatal development of retinocollicular projections in the cat: An anterograde tracer transport study. J Neurosci 2:604.

Williams RW, Chalupa LM (1982b) The effects of prenatal unilateral enucleation upon the functional organization of the cat’s lateral geniculate nucleus. The Physiologist 25:223.

Williams RW (1983) Prenatal Development of the Cat’s Visual System. Thesis, University of California, Davis.

Williams RW, Chalupa LM (1983a) Development of the retinal pathway to the pretectum of the cat. Neuroscience, in press.

Williams RW, Chalupa LM (1983b) Expanded retinogeniculate projections in the cat following prenatal unilateral enucleation: Functional and anatomical analyses of an anomalous input. Soc Neurosci Abstr 9: in press.

Williams RW, Bastiani MJ, Chalupa LM (1983a) Addition and attrition of axons within the optic nerve during fetal development: Appearance of growth cones and necrotic axons. Invest Ophthalmol Vis Sci Suppl 24:8.

Williams RW, Bastiani MJ, Chalupa LM (1983b) Loss of axons in the cat optic nerve following fetal unilateral enucleation: An electron microscopic analysis. J Neurosci 3:133.


 

 


   


Neurogenetics at University of Tennessee Health Science Center

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