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Note to the Reader This is a revised version of a paper published in The Journal of Comparative in 1986. Several figures have been added. Additions are delimited by brackets [...].
Enlarging images: Thumbnail versions of all figures are embedded in the paper. A better image—usually under 100K—will download into a separate window if you click on the thumbnail image. If you have a large enough monitor drag this second figures window beside the text window. Finally, high-resolution images—usually under 600K—that almost match the quality of the original prints can be downloaded by selecting the text at the bottom of each legend. These image files can be viewed with Adobe Photoshop, NIH Image, or equivalent.
Revised HTML edition (http://www.nervenet.org/papers/cat86.html) copyright © 1998 by Robert W. Williams.

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Growth Cones, Dying Axons, and Developmental Fluctuations in the Fiber Population of the Cat’s Optic Nerve

Robert W. Williams, Michael J. Bastiani, Barry Lia, Leo M. Chalupa
Department of Psychology and Physiology Graduate Group, University of California, Davis, California 95616 (R.W.W., B.L., L.M.C.) and Department of Biological Sciences, Stanford University, Stanford, California 94305 (M.J.B.)
 

The Journal of Comparative Neurology 246:32–69 (1986)

            

Table of contents


ABSTRACT

We have studied the rise and fall in the number of axons in the optic nerve of fetal and neonatal cats in relation to changes in the ultrastructure of fibers, and in particular, to the characteristics and spatio-temporal distribution of growth cones and necrotic axons.

Fiber number. Axons of retinal ganglion cells start to grow through the optic nerve on the 19th day of embryonic development (E19). As early as E23 there are 8,000 fibers in the nerve close to the eye. Fibers are added to the nerve at a rate of approximately 50,000 per day from E28 until E39—the age at which the peak population of 600,000 to 700,000 axons is reached. Thereafter, the number decreases rapidly: About 400,000 axons are lost between E39 and E53. In contrast, from E56 until the second week after birth the number of axons decreases at a slow rate. Even as late as postnatal day 12 (P12) the nerve contains an excess of up to 100,000 fibers. The final number of fibers—140,000 to 165,000—is reached by the 6th week after birth.

Growth cones of retinal ganglion cells are present in the optic nerve from E19 until E39. At E19 and E23 they have comparatively simple shapes but in older fetuses they are larger and their shapes are more elaborate. As early as E28 many growth cones have lamellipodia that extend outward from the core region as far as 10 µm. These sheet-like processes are insinuated between bundles of axons and commonly contact 10 to 20 neighboring fibers in single transverse sections. At E28 growth cones make up 2.0% of the fiber population; at E33 they make up about 1.0%; from E-36 to E39 they make up only 0.3% of the population. Virtually none are present in the midorbital part of the nerve on or after E44. At all ages growth cones are more common at the periphery of the nerve than at its center. This central-to-peripheral gradient increases with age: at E28 the density of growth cones is two times greater at the edge than at the center but by E39 the density is 4 to 5 times greater.

Necrotic fibers are observed as early as E28 in all parts of the nerve. Their axoplasm is dark and mottled and often contains dense vesiculated structures. From E28 to E39 an average of about 0.15% of all fibers are obviously necrotic, whereas during the most acute phase of fiber elimination—between E44 and E48—up to 0.4% are necrotic. Thereafter, their incidence is typically under 0.05%. Necrotic axons are scattered throughout the nerve. We estimate that the time required to clear away the debris of single axons is short—on the order of 1 hour—and based upon this estimate, we conclude that between 100,000 and 200,000 axons are lost even before the peak population of 700,000 is reached.

Taking into account the early loss of fibers, we estimate that a total of 800,000 to 900,000 retinal ganglion cell axons are produced in the fetal cat over a 20-day period from E19 to E39. Remarkably, only 20% survive to adulthood. The loss of fibers begins a few days before axons penetrate the thalamus (Shatz, 1983), about two weeks before the onset of synaptogenesis in the dorsal lateral geniculate nucleus (Shatz and Kirkwood, 1984), and more than three weeks before the segregation of the retinal projections (Williams and Chalupa, 1982, 1983a; Shatz, 1983; Chalupa and Williams, 1984). The elimination of axons also persists long after the segregation of axon arbors from right and left eyes is complete, and as many as 100,000 axons are lost even after eye opening during a one month period when retinal arbors are still undergoing remarkable changes in shape and connectivity (Mason, 1982a,b; Sur et al., 1984).

[Key words: axon necrosis, axon number, neuron death, neuron number, retinal ganglion cells, retinal projections]


 

INTRODUCTION

A great excess of axons are produced and subsequently lost during the early development of the avian and mammalian optic nerve. Although there have been numerous quantitative studies of the nerve during development, we still know little about the relationship between this rise and fall in axon number and the corresponding changes in the nerve’s ultrastructure (Rager and Rager, 1976; Rager, 1980; Ng and Stone, 1982; Rakic and Riley, 1983a; Perry et al., 1983; van Driel and Provis, 1983; Crespo et al., 1984; Sefton and Lam, 1984; Kirby and Wilson, 1984;). Our main aim has been to fill this gap: to provide a complete description of the maturation of the optic nerve—both qualitative and quantitative—and to answer the following specific questions:

• When are growth cones present in the nerve and what are their characteristics? The resolution of these two questions, which are of broad relevance to issues of axon elongation, requires a detailed analysis of growth cone ultrastructure, number, and distribution within the nerve throughout development.

• When are axons eliminated from the nerve and what are the signs of axon elimination? Although it is clear that a large proportion of axons are lost spontaneously during normal development, almost nothing is known about the ultrastructure, distribution, or timing of axon elimination.

• What is the total number of axons produced during the formation of the optic nerve? It is generally assumed that the total production of axons equals the peak number of axons in the nerve, but this assumption is not valid if proliferative and degenerative phases of development overlap; that is, if there are both growth cones and necrotic axons in the nerve at the same time. By obtaining information on the duration of overlap of axon ingrowth and necrosis it should be possible to estimate the degree to which the peak axon number underestimates total fiber production.

We have chosen to study the cat’s optic nerve because so much is known about the genesis and maturation of this species’ retina (Martin, 1891; Donovan, 1966; Cragg, 1975; Morrison, 1975, 1982; Rusoff and Dubin, 1977; Vogel, 1978; Greiner and Weidman, 1980; Polley et al., 1981; Stone et al., 1982; Rapaport and Stone, 1982, 1983; Lia et al., 1983; Walsh et al., 1983; Mastronarde et al., 1984; Walsh and Polley, 1985), and because the sequence of retinal innervation of the cat’s dorsal lateral geniculate nucleus, pretectum, and superior colliculus has been well studied (Cragg, 1975; Winfield et al., 1980; Williams and Chalupa, 1982, 1983a; Mason, 1982a,b; Shatz, 1983; Sretavan and Shatz, 1984; Chalupa and Williams, 1985). This rich background provides an opportunity to relate changes in the fiber population of the optic nerve with the development of the visual system.

 

MATERIAL AND METHODS

Animals and the determination of gestational age

This study is based on an analysis of 19 optic nerves taken from cats ranging in age from the 19th day of gestation (E19) to the end of the third postnatal month. Litters of known gestational age were obtained by placing an estrous female together with a tomcat for 24 h. Ovulation in cats occurs 24 to 30 h after mating (Greulich, 1934; Herron and Sis, 1974) and the ova are viable for an additional 24 h (Hoogeweg and Folkers, 1970). Fertilization therefore occurs the day after mating—embryonic day 1 or E1. In our colony most cats give birth within a day or two of E65. However, viable offspring can be born as early as E58 or as late as E70 (Marin-Padilla, 1971; Prescott, 1973; Stein, 1975). [Eye weight data (figure A) were obtained from most fetal cats to assess rates of eye growth and to provide an independent morphological measure of the developmental stage of a litter or animal.]

Figure A

Figure A. Growth of the eye of the cat duing the second half of gestation. The Y axis is scaled logarithmically. Eye weight increases ten-fold between E28/30 and E34/35. Between E35 and P10 (eye opening) the eye grows at a fairly constant exponential rate. Single adult eyes weigh approximately 5 gm.


 

 

Surgical and histological procedures

Pregnant females were anesthetized with 1.5% Halothane in oxygen or by an intravenous infusion of sodium pentobarbital. Incisions were made through the abdomen and uterus, and fetuses were removed one at a time and perfused immediately through the heart with 5–10 ml of saline followed by a fixative made up of 2% glutaraldehyde, 1% paraformaldehyde, 1% dimethyl sulfoxide, and 5 mM magnesium chloride in 0.05 M sodium phosphate buffer (pH of 7.4 ± 0.1) used at room temperature. In the youngest embryos (E19 and E23) with crown-to-rump lengths of 12–15 mm the saline rinse was omitted and the perfusion was begun immediately with fixative at a pressure of approximately 50–60 cm of water. The fixative was also injected behind the eye into the orbit. Postnatal animals were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital and perfused transcardially as above.

Eyes and optic nerves were dissected in cold buffer. The dural sheath was removed gently, and the nerves were cut into short segments. Those pieces chosen for analysis were from the orbital portion of the nerve and were usually taken 1.0 to 3.0 mm from the eye. The eyes and nerves that were removed from the youngest animals, E19 and E23, were left intact. Following a wash in buffer, tissue was placed in a solution of 2% osmium tetroxide for 1 h, stained with 2% aqueous uranyl acetate, dehydrated, and embedded in Epon-Araldite. Semithin sections were cut at 1.0 µm for light microscopy and ultrathin sections were cut at about 0.08 µm for electron microscopy. Ultrathin sections were mounted on Formvar-coated slot-grids or on uncoated 400-mesh grids and stained with uranyl acetate and lead citrate. The 1-µm-thick sections were stained with a mixture of Azur II and methylene blue.

Sampling, measuring, and counting of fibers

Except at the earliest stage of development it was not practical to count all fibers in the optic nerve. Instead an estimate of the total number was made on the basis of the average density of axons in a representative set of micrographs. To obtain reliable and accurate estimates we employed without modification a procedure described in our previous studies (Williams et al., 1983; Williams and Chalupa, 1983b). Micrographs intended for counting were taken with Zeiss or Hitachi electron microscopes at instrumental magnifications ranging from 1,400 to 12,000. The sampled sites were distributed with as much uniformity as possible across the entire section of the nerve, and therefore, each region of the nerve was represented in proportion to its contribution to the total area of the transverse section. Exposures were also taken without regard for the particular elements, be they axonal, glial, or vascular, that happened to dominate the field of view.

A variety of sampling strategies have been employed to estimate the number of axons in the optic nerve, including simple random sampling (Rhoades et al., 1979), sampling along two or more diameters (Rakic and Riley, 1983a), sampling each major fascicle (Easter et al., 1981), and sampling systematically (Vaney and Hughes, 1977). We chose to sample systematically using the square grid method (Cochran, 1963, p. 229), both because this technique provides a simple and economical way to get a representative sample, and because such systematic samples usually give estimates with less variance than do random samples of corresponding size (Cochran, 1963, pp. 223–229). It is important to recognize that within wide limits, the accuracy of the method we employed depends far more on the number of sampled sites than upon the percentage of the area that is sampled (see Snedecor and Cochran, 1967, p. 513). Therefore, no attempt was made to sample equal proportions of the nerves. In several sections obtained from the youngest embryos (E19 and E23) it was possible to make complete high magnification (×8,000) montages and count all axons.

Calculation of fiber number. To estimate the number of axons in the optic nerve three values were determined: (1) the total area of the nerve in the particular ultrathin section that was photographed, (2) the area of nerve covered by the sample of micrographs, and (3) the number of axons within the area that was sampled. An estimate of the total population was then calculated by multiplying the number of axons that were counted by the ratio of the nerve area and the sampled area.

Accurate measurements of area were obtained using a calibration grid (0.214 µm2/grid unit, specified as accurate to within 0.05%, Ernest F. Fullam, Inc., U.S.A.). Areal magnification—the square of linear magnification— then determined by counting the total number of grids covered by the calibration micrographs, and the square root of this value was used to calculate the mean linear magnification. This value was used to calculate the area of individual micrographs and of low-power photomontages of the nerves.

The number of axons in each micrograph was determined using Gundersen’s rule (1977). His method is slightly more accurate than that usually employed to correct for the discrepancy between the effective sampling area and the actual micrograph area. This discrepancy, termed the edge effect, arises when axons of which only small parts are within the margins of the micrograph are nevertheless counted. If uncorrected, the inclusion of these marginal fibers leads to an effective sample area greater than the actual micrograph area. The correction involved excluding from the count all axons that intersected the lower or left edges, or that intersected any corner other than the upper right corner. All counts were checked twice.

Accuracy of estimates. The accuracy of estimates of axon number depends upon two factors: (1) the sample size and its spatial resolution in relation to gradients of axon density, and (2) the accuracy of the count and of the measurements of area. The adequacy of the sample can be tested by breaking the pool of micrographs into subgroups and using these to calculate a number of ‘minority’ estimates (Williams et al., 1983). Four nerves were tested using this procedure and the mean divergence of these estimates was less than 5%. We also determined the reliability of our sampling method by estimating the axon complement within two ultrathin sections cut from either side of a 1-mm-long segment of the nerve. The sections were photographed using different microscopes (Zeiss EM109 and Zeiss 10), and prints were made on different enlargers. Procedural details, however, were identical. Final estimates differed by less than 4% (Table 1).

Measurement of fiber caliber. The area and perimeter of 2,000 fibers in each nerve were measured using an image analysis system (Zeiss Videoplan), and the diameter of a circle with an area equivalent to that of each fiber was calculated and used to make histograms. Each histogram represents a uniform and unbiased sample of the transverse section. Profiles of large axons and growth cones are more likely to intersect the boundaries of micrographs than are those of small axons, and consequently their contribution and size will tend to be underestimated. To sidestep this source of error we placed a mask with a wide border over micrographs and measured the area of all axons completely within the central hole of the mask. The mask was then removed and the areas of those fibers that had initially intersected the margins of the mask were measured.


 

            
 

RESULTS

As is true of other parts of the central nervous system, the 12–18 mm long optic nerve of the adult cat contains oligodendrocytes, astrocytes, and blood vessels, and is surrounded by a glial limiting membrane, a basal lamina, and meninges. In the adult cat there are 140,000 to 165,000 retinal ganglion cells in each eye (Illing and Wässle; 1981; Chalupa et al., 1984) and a corresponding number of fibers in each optic nerve (Williams et al., 1983; Williams and Chalupa, 1983b; Chalupa et al., 1984; Williams et al., 1985).

The results are described in three sections. The first deals with quantitative aspects of nerve development; the second describes the ultrastructure of the nerve during axon ingrowth, concentrating on the characteristics of ganglion cell growth cones; the third section summarizes our findings on fiber necrosis.

 

Quantitative aspects of nerve development

Rise and fall in fiber number. We determined the number of fibers in the optic nerve at 13 prenatal and 4 early postnatal ages (Table 1, Fig. 1). The word fiber is used here to include normal axons, growth cones, and necrotic axons. Axons of retinal ganglion cells enter the precursor of the optic nerve—the optic stalk—at the end of the 3rd week of gestation. However, merely 88 fibers were counted in a midorbital section of the optic stalk taken from an E19 embryo (Figs. 3, 4). But by E23 there were nearly 100 times as many axons in a section of the nerve taken close to the eye, although remarkably, a section of the same nerve taken near the optic chiasm (Fig. 6) contained merely 8 fibers, 5 of which were growth cones! The striking difference between these two sections indicates that few if any fibers had yet grown as far as the chiasm.

Figure 1
 

Fig. 1. Number of fibers in the optic nerve during development. Individual data points (see Table 1, below) are plotted as circles. The peak fiber number (560,000 to 700,000), reached at around E39, underestimates total axon production by 100,000 to 200,000 because numerous axons are lost before E39. The gray stripe above the peak therefore represents the approximate total axon production. The apparent rise in axon number from E52 to P2 is a sampling artifact. Fiber number in individual nerves either actually decreases slightly during this time as demonstrated by the small number of necrotic axons in nerves during the perinatal period. The adult value is reached as early as the 100th day after conception or about 36 days after birth.


Table 1. Size and Character of the Axon Population of the Optic Nerve during Development


      Area
of Nerve
Axon Density Necrotic Fibers Growth Cones
AGE N Axons ±SEM µm2 100 µm2 % %

E19 88   2,300 3.8 0
E23¶ 8,000 ± 1,500 3,800 210
E28 43,000 ± 3,000 13,000 330 0.23 2.0
E33 292,000 ± 12,000 43,900 665 0.09 1.0
E36 490,000 ± 28,000 76,500 645 0.10 0.3
E39* 557,000 ± 28,000 74,400 749 0.21 0.3
E39* 698,000 ± 20,000 95,700 729 0.26 0.2
E44** 580,000 ± 13,000 93,000 623 0.41 >0.05
E44** 457,000 ± 21,000 88,100 518 0.40 0
E47 403,000 ± 31,000 124,000 325 0.15 0
E48 328,000 ± 17,500 148,300 221 0.28 0
E52 308,000 ± 30,000 179,500 172 0
E53 225,000 ± 18,000 177,000 127 >0.05 0
E56 230,000 ± 21,000 157,800 146 >0.05 0
E61 267,000 ± 18,900 331,000 81 >0.05 0
P2 293,000 ± 19,400 442,900 89 >0.05 0
P12 250,000 ± 6,200 494,800 51 0.19 0
P36 158,000 ± 9,500 1,200,000 13 0.22 0
P84 162,000 ± 8,300 1,025,000 15 0.05 0

§ To calculate standard error of the mean we first calculated the standard deviation of the number of axons in the set of micrographs from a given nerve. The standard deviation was then divided by the square root of the number of micrographs (minus 1) to give the error of the average number of axons per micrograph. This error term was multiplied by the ratio between the total area of the ultrathin section and the sample area that yielded the standard error of the mean. Any systematic regional variation in the density of axon packing, as in the optic nerve of adult cat’s, would obviously result in an overestimate of the standard error calculated as described above. But we found that the packing density of fibers in the fetal optic nerve was comparatively homogenous and for this reason the simple formula we have used is reasonably accurate.
¶ Estimate from this animal is accurate only to within ± 20% due to angle of section.
‡ Not analyzed.
† Percentage of growth cones depends on distance from the eye. Range from 2% to 100%.
*  Littermates
** Littermates


By E28, 43,000 fibers had extended into the orbital part of the nerve (Table 1, Fig. 16). During the next five days the number of fibers increased rapidly—nearly 50,000 axons were added each day, and already by E33 the nerve contained 292,000 axons, about twice as many axons as in the mature nerve. The number of fibers and their density of packing reached a peak at E39, nearly three weeks after the entrance of the first axons (Table 1): as many as 698,000 fibers were packed together at a density of 7.5 per 1.0 µm2, twice the value at E28 and approximately 100 times the adult value (Table 1). This high population was largely retained until E44: 2 nerves of littermates at E44 contained 580,000 and 454,000 fibers. The substantial difference in the number of fibers—up to 23%—in nerves from littermates at E44 and at E39 (Table 1) concerned us. Was it real or did it result from inaccurate methods? To solve this problem a second ultrathin section was cut from the same E44 nerve that we had estimated contained 457,000 ± 21,000 fibers. This second section, located 1 mm closer to the chiasm, contained 441,000 ± 28,000 fibers. The close agreement between these estimates indicates that the differences between littermates reflects individual variation rather than technical variability.

Figure 2
 

Fig. 2. Histograms of fiber size in the prenatal optic nerve of the cat. At E28 (A) large axons contribute to the right-side tail of the histograms. The bin to the far right of histograms A, B, and C represent growth cones with diameters above 1 µm. As early as E33 the contribution of large axons is less pronounced (B), and as growth cones extend beyond the optic nerve at E39 and E44 (C, D) the large axon component disappears and as a consequence the average fiber diameter drops to about 0.3 µm. After E48 (E, F), the mean diameter increases steadily reaching about a third the adult value when myelination begins at around birth. All histograms are based on 2000 measurements distributed evenly across cross-sections of nerves. In E and F the small overflow bins simply represent large axons.

The number of axons in the nerve dropped sharply between E44 and E53. Indeed, it was reduced to less than half its peak value: a nerve from an E47 fetus contained 403,000 fibers, that from an E48 fetus contained 328,000 fibers, that from an E52 fetus contained 308,000 fibers, and that from an E53 fetus contained 225,000 fibers (Table 1).

During the perinatal period, from E56 through P12, no consistent downward trend in axon number was evident: for example, a nerve from an E56 fetus had as few as 230,000 fibers, whereas a nerve from a 3-day-old kitten had 293,000 fibers. However, given our results on the incidence of necrotic fibers in the nerve during this period (described below), it is quite likely that the axon population in individual nerves did, in fact, decrease at a slow rate. By P36 the number of axons in the nerve had reached a mature value of 158,000; within the adult range we have encountered in normal adult cats (Williams et al., 1983; Williams and Chalupa, 1983b; Chalupa et al., 1984; Williams et al., 1985).

Figure 3

Fig. 3. The optic stalk—precursor of the optic nerve—at E19. This section contains 86 axons and two growth cones (see Figure 46). Most fibers are located in the lower, ventral part of the stalk in prominent extracellular ducts. Arrowhead marks the site of the growth cone reproduced in Figure 6A. Only a few axons are situated in the upper half of the stalk (arrows mark the axons shown in Fig. 5A and B. Necrotic cells, characterized by dispersed ribosomes, ruptured nuclei, and dark, mottled cytoplasm, are prominent at the 7, 9, and 11 o’clock positions. Processes of several necrotic cell partly fill ducts. At this age the lumen of the stalk (center) is still patent. Cells in the upper left portion of the figure extend radially the full width of the tube. In contrast, cells in the ventral portion of the stalk are do not have a radial orientation. Calibration bar is 10 µm. Download the high-resolution 544 KB image.

The growth of axons in caliber. In the optic nerve of the adult cat there is large variation in the caliber of the myelinated fibers; the smallest axons are 0.2 µm in diameter (excluding the myelin sheath), the largest are 7.5 µm in diameter, but the overall distribution of fiber size in the mature nerve is bimodal with modes at about 1 and 2 µm (Williams and Chalupa, 1983b; Williams et al., 1985). Fibers are packed together at a mean density of 7 to 9/100 µm2. In contrast, in the fetal cat all axons are unmyelinated, the distribution is strictly unimodal, and axons are packed together at a density which at its peak is 100 times greater than in the adult nerve!

Based on histograms of axon caliber, the growth of optic fibers in the fetal cat was divided into three stages (Fig. 2). The first stage lasted from the onset of axon ingrowth until about E28. The most striking feature of this period was that the nerve contained many large axons that made up a sizable fraction of the total fiber population and that contributed to an extensive histogram tail (Fig. 2A). These large axons are actually the long trailing part of the fiber located just behind the growth cone, and as expected, the number of such large fibers at early ages is related to the number of growth cones. For instance, at E28, 10% of all fibers have diameters above 0.6 µm and 2% of all fibers are growth cones with diameters above 1.1 µm (see below). By E33 only 4% of axons are larger than 0.6 µm in diameter and there is a corresponding drop in the density of growth cones to about 1.0%.

Figure 4

Fig. 4. Distribution of the first 88 fibers in the mid-orbital part of the optic stalk at E19. The positions of axons are marked by small dots, and of two growth cones by stars on the right side.

The second period started as early as E33 and lasted until E48. Growth cones and large axons, although still present in the nerve up until E39, made up a comparatively small fraction of the total fiber population. The decrease in the fraction of growth cones in the nerve led to a corresponding drop in the range and average size of fibers; mean fiber diameter decreased from 0.37 µm at E28 to approximately 0.30 µm between E33 to E44 (Fig. 2B, C, D , E). The histograms also became more nearly symmetrical about their modes because of this loss of the rightward extending ‘growth cone’ tail. The surprising feature of the second period was that there was no increase in fiber diameter: during this period, axons grew exclusively in length. However, even as early as E44, before cumulative histograms of fiber diameter display any noticeable upward shift in axon diameter, there are both isolated instances of large axons and even a few collections of large axons (for example, those in the upper half of Figure 22). Some of these may be the trailing ends of growth cones, but at least a fraction may simply be large caliber axons.

Figure 5

Fig. 5. The first axons in the optic stalk at E19 (A) Single isolated axon situated between radially oriented neuroepithelial cell processes in the optic stalk 12.5 µm from the edge of the nerve and about 11 µm from the lumen and the nearest axon. The site is marked by an arrowhead in Figure 3.(B) Fascicle of two axons both of which contain neurofilaments. The loose vesicular material within the duct (arrowhead) may be the remnants of a growth cone ruptured during fixation. Diameters of these axons are 0.39 and 0.46 µm. (C) A tightly packed fascicle of 11 axons at the ventral periphery of the stalk. The large fiber that contains an abundance of tubulo-vesicular material and several neurofilaments is probably sectioned close to, or through, the core region of the growth cone. All calibration bars are 1 µm. Download the high-resolution 400 KB image.

The third period of growth began as early as E48, roughly concurrent with the onset of segregation in the lateral geniculate nucleus (Shatz, 1984; Chalupa and Williams, 1985). At this age the diameter of axons began to increase steadily. Although the minimum diameter of optic axons remained about 0.2 µm, the range increased considerably, peak values reaching up to 1 µm (Fig. 2F, G, H). No myelinated axons were present in the nerve at E56, but by E61 a small number of fibers were ensheathed by broad glial tongues (Fig. 24), and an even smaller number of fibers (96 out of 9,140) were already surrounded by thin rims of compacted myelin. Three days after birth the nerve differed only in that the number of myelinated fibers and the thickness of their myelin was greater. By P12 the nerve was much maturer in appearance (Fig. 25A); about 25% of the axons were myelinated, and another 30% were pro-myelinated axons in the process of receiving their first glial wraps. Associated with the onset of myelination, the optic fibers grew substantially; diameter increased rapidly from 0.5 µm at P2 to 0.7 µm at P12, and by P84 mean axon diameter had already reached 1.7 µm; close to typical adult values (Williams and Chalupa, 1983b). However, even as late as P84 the bimodal distribution was not as pronounced as in adults.

Figure 6

Fig. 6. Growth cones and neuroepithelial processes at E19 and E23. (A) Growth cone at E19 marked by arrowhead in Figure 3. The first growth cones in the nerve are large, pale and generally had few and very simple lamellipodia. This growth cone has a diameter of 1.6 µm and in this section contains no neurofilaments. (B) Growth cone at E23 with a diameter of 2.3 µm. Although this growth cone resembles that reproduced in A, it contains many neurofilaments and a large network of endoplasmic reticulum. Arrowhead marks a coated vesicle.(C) Three-fiber fascicle. The diameters of these fibers are 1.6, 1.5, and 1.1 µm. The larger fibers are sectioned at or near the core of the growth cone (see text). A cluster of neurofilaments in the lower growth cone is marked by an arrowhead. (D) Neuroepithelial processes apposed to the basal lamina. Although it resembles a large growth cone, it contains many ribosomes (see INSET magnified 3-fold), rough endoplasmic reticulum, and comparatively large mitochondria and thus is actually a neuroepithelial endfoot. Calibration bars are 1 µm. Download the high-resolution 425 KB image.

Size and organization of fascicles. At E28 each of the 100 fascicles in the nerve contained about 400 fibers. By E33 the number of fascicles had increased to nearly 300, and each of these contained an average of 1,000 fibers (Fig. 15). Fascicles probably fuse with one another and split apart a great deal in the cat, as they do in monkey (Williams and Rakic, 1985a) and mouse (Silver, 1984), and thus the precise number of fascicles is likely to vary along the length of the nerve. Variation in the size of fascicles was substantial: the smallest contained 10 to 100 fibers, and the largest contained more than 2,500 fibers (Figs. 7–9). The 7-fold increase in the total fiber population between E28 and E33 was associated with 3-fold increase in the number of fascicles and a 2.5-fold increase in the number of axons per fascicle. Both central and peripheral fascicles grew considerably in size between E28 and E33, and the size of fascicles was not strongly related to their eccentricity at any age. This suggests that new fascicles were not simply added at the periphery of the nerve as successive waves of axons grew into nerve, because if this were the case, central fascicles would probably retain a relatively stable population of axons and the newest fascicles at the extreme periphery would be comparatively small.

Despite a 2.4-fold increase in the number of fibers between E33 and E39, the average number of fibers per fascicle rose merely 8%—from 1,000 to about 1,080. Naturally, the increase in the population of fibers was accompanied by a substantial increase in the number of fascicles. In comparison to the E33 nerve that contained 289 fascicles, the E39 nerve with the largest axon population contained 550 distinct fascicles, each set off from its neighbors by a glial partition 0.2 to 2.0-µm-thick. Since new fibers grew into virtually all fascicles even as late as E33 (see the following section), the constant fiber population per fascicle between E33 and E39 suggests that the maximum size of fascicles in the nerve is regulated in some manner by glial cells and their processes, and that fascicles are repartitioned throughout the period of axon ingrowth.

Figure 7c

Fig. 7. Axons and growth cones at E28. (A) Fascicle at the nerve’s periphery. Two growth cones with perimeters of 12 and 16 µm are sectioned through their lamellipodia (large asterisks). Another three growth cones (small asterisks) are sectioned through their cores. The remaining fibers in this fascicle, although not categorized as growth cones, are remarkably large (mean diameter is 1 µm) and contain many microtubules and neurofilaments. Large axons are probably cut close to their expanded tips. Note the difference in size of mitochondria in glial cells and in fibers and growth cones. Two electron-dense junctions between adjacent cells of the glial limiting membrane are marked by arrows at the edge of the nerve. (B) Central fascicles of axons at E28. The 22 axons in the very small central fascicle have an average diameter of 0.39 µm, about one-third the value of those in A. This field contains a single growth cone (asterisk) sectioned through or near the core. Axons in central fascicles are smaller and probably older. Neurofilaments tend to cluster close to the edges of fibers. One axon (arrowhead) contains a coated vesicle. As at the periphery, processes of glial cells are occasionally linked together by small junctions (bent arrow). Very large coated pits were common on glial cells (arrows). Calibration bar is 1 µm.

Two extremely high resolution scans of orginal prints of Fig 7A and 7B represent the best image quality avaiable with this WWW edition. Both images surpass the print publication reproductions:

Starting at E44 the processes of glial cells extended into and subdivided axon fascicles (Fig. 23). Because fascicles were not as distinct as at earlier ages their number could only be approximated. Nerves taken from animals between E44 and E53 had 400–500 major fascicles (about the same number as at E39), but each of these was split into 2, and in some cases, as many as 8 smaller compartments (Fig. 23). Glial proliferation and growth continued vigorously as late as P12, and the mixture between glia and groups of axons was so thorough during the perinatal period that it was not even possible to approximate the number of fascicles.

Figure 7c

Fig. 7c. Axons and growth cones at E28. Growth cones have been tinted orange, whereas axons with diameters about 0.75 µm have been tinted yellow (and also marked with asterisks). Glial processes bisect the field and are tinted blue. Calibration 1 µm. Download a very high-resolution 400 KB image.


 

 

Ultrastructure of the nerve during axon ingrowth

Optic stalk. From E19 until at least E23, the precursor of the optic nerve—the optic stalk—consisted largely of neuroepithelial cells. Some of these cells were columnar and spanned the full width of the wall of the stalk (upper right quadrant of Figure 3). Other cells appeared to have lost their inner, lumenal processes and to have moved toward the periphery of the stalk through which the first axons grow (see especially the lower half of Figure 3). However, as early as E23, the radial arrangement of neuroepithelial cells was no longer apparent. Adjacent lumenal (ventricular) processes of neuroepithelial cells were joined together by intermediate junctions up to 2 µm long. Their most characteristic feature was an extremely dense staining of the cytoplasm just beneath the cell membrane. In contrast, the peripheral ends of neuroepithelial cells were joined sporadically by small junctions that resembled, but were distinct from, puntae adherens described previously in a classic study of the meninges by Nabeshima and colleagues (1975, p. 131, their figures 21 and 22). Frequently however, no junctional specializations of any type were evident between the marginal ends of neuroepithelial cells (e.g., Fig. 5B).

One prominent feature of the optic stalk at E19 and E23 was the abundance of large, roughly circular intercellular spaces between the peripheral ends of neighboring neuroepithelial processes (Fig. 3). These spaces ranged in size from 0.5 to 6 µm, but typically had diameters of about 2 µm. Approximately 140 were counted in transverse sections through the E19 stalk. The majority of these spaces or ducts did not contain any fibers or other cell processes, and ganglion cell fibers grew within an apparently undistinguished subset of ducts. Similar large intercellular spaces—or intercellular lakes to use the phrase of Silver and Robb (1979)—are prominent in the eye during early stages of axon elongation, and it has been argued that these spaces actually form an aligned system of ducts that guide or polarize the growth of axons (von Szily, 1912; Ulshafer and Clavert, 1979; Silver and Robb, 1979; Krayanek and Goldberg, 1981). More recently, it has been recognized that the channels actually form a maze rather than as an orderly linear array (Suburo, 1979; Silver and Sapiro, 1981), and it therefore seems that their role with respect to fiber growth is permissive—not instructive.

Figure 8

Fig. 8. Axons and growth cones in a peripheral fascicle at E33. This fascicle contains 752 fibers with an average diameter of 0.30 µm. Most growth cones (asterisks) have diameters greater than 1.3 µm. Growth cones with long lamellipodia are prominent in the lower, peripheral part of the fascicle. One growth cone with elaborate lamellipodia has a perimeter of 25 µm (star) and a total of 58 neighboring fibers. The core region of a large growth cone (arrow) with a diameter of 1.7 µm contains a labyrinthine smooth endoplasmic reticulum (see Figure 11). Vesicular aggregates in both growth cones and axons are marked by arrowheads. Glial cells occupy almost precisely 25% of the nerve at this age. Their cytoplasm is dense and contains numerous ribosomes and is easily distinguished from axons and growth cones. No glial processes intrude into the fascicle itself. The nerve is still entirely avascular at this age. Calibration bar is 1 µm. Download the high-resolution 300 KB image.

Many cells of the stalk were necrotic at E19 and E23. In the single section reproduced in Figure 3, several dying cells are visible, and when one takes into account the short duration of necrosis—on the order of 3 hours (Glücksmann, 1951; Hughes, 1961; Senglaub and Finlay, 1982)—it seems probable that a large proportion of cells in the stalk die over a short period. The appearance of these dying cells varied—some contained many lysosomes, autophagic vacuoles, heavily condensed chromatin or completely obliterated nuclei, and dense aggregates of dark, undefinable debris (compare with Chu-Wang and Oppenheim, 1978a). Others contained less debris and had comparatively pale cytoplasm with dispersed ribosomes. In several cases normal neuroepithelial cells had sequestered debris of necrotic cells in large phagosomes.

Figure 9

Fig. 9. Central fascicle at E33 that contains 685 axons and 3 growth cones. Three growth cones are situated in the central part of this fascicle and at this level lack contact with glial cells. Calibration bar is 1 µm. Download the high-resolution 300 KB image.

Von Szily (1912) demonstrated that a wave of cell necrosis sweeps through the optic stalk from the retina toward the brain in advance of the ingrowth of the optic axons. In our tissue several of the duct in fact do appear to contain necrotic processes (Fig. 3) and for this reason we find von Szily’s idea that the ducts are just holes left behind by dying cells plausible. However, whether, as von Szily suggested (p.84), ingrowing axons are attracted to necrotic debris and whether the wave of necrosis serves to guide axon elongation remains controversial, especially in view of the fact that necrosis is apparently rare in the optic stalk of Xenopus (Cima and Grant, 1982).


Figure 10

Fig. 10. Longitudinal section of axons and growth cones at E33. The edge of the nerve is shown in the lower left corner. The large growth cone in the middle of the field (asterisk) is at least 25 µm long. Microtubules in this growth cone are present to the left of the asterisk. The growth cone is probably extending in the direction of the large arrow. Coated pits (arrowhead in the upper-right quadrant), coated vesicles (arrowhead), and a dark core vesicle (small arrow) are common in axons as well as growth cones. Calibration bar is 1 µm. Download the high-resolution 434 KB image.


 

Ultrastructure of axons. The first axons in the optic stalk contained from 3 to 10 microtubules, clear vesicles, irregular membrane profiles (probably cross-sections of the smooth endoplasmic reticulum), and only limited amounts of microfilamentous material (Fig. 5). In comparison to fibers in older fetuses, the axoplasm of the first complement of fibers was only lightly stained (compare Fig. 5 with Fig. 24), in large measure because of the low concentration of microfilaments and neurofilament. Although points of contact between neighboring axons and between axons and neuroepithelial processes were common, we saw no evidence of membrane specializations, either gap junctions or desmosomes.

Figure 11

Fig. 11. Tubulo-vesicular mazes, probably part of the smooth endoplasmic reticulum, enmeshed in neurofilaments at E33. These structures are common in the core region of growth cones (also see Figure 7). Calibration 1 µm.

At E19,fibers were distributed around the whole perimeter of the stalk (Fig. 4). Nonetheless, as in other vertebrates (Müller, 1874; Robinson, 1896; Silver and Sapiro, 1981), most axons were located within the ventral half of the stalk and less than 5 µm from the outer margin. The mean distance between fibers and the edge of the optic stalk was merely 2.7 µm. Although the young optic axons were very close to the margins of the central nervous system, none were actually seen at the extreme periphery of the stalk, apposed to the basal lamina at either E19 or E23, or for that matter, at any later stage of development. Although several processes with pale cytoplasm similar to growth cones were occasionally seen on the basal lamina (Fig. 6D), in every case when examined at high magnification they were found to contain numerous ribosomes and polysomes (Inset to Fig. 6D) and were therefore actually endfeet of neuroepithelial cells. At E19, 10 axons were located farther than 5 µm from the edge of the nerve, and in one extreme case, an axon was situated almost at the center of the stalk within 1 µm of the lumen.

Isolated, small caliber axons were found in the nerve at both E19 and E23 (Fig. 5A). The extension of growth cones is therefore almost certainly not contingent upon their proximity to other axonal surfaces. Similarly, the existence of axons deep within the wall of the stalk far from any other axons indicates that growth cones are able to penetrate between neuroepithelial cells and that they do not require contact with, or even proximity to, the endfeet of neuroepithelial cells. This result should be contrasted to the growth of fibers in the optic stalk of Xenopus in which it has been shown that axons are rarely if ever seen in isolation (Cima and Grant, 1980, p. 232).

Figure 12
 

Fig. 12. Deep penetration of a growth cone (left) by a glial process (right) at E33. A small uncoated vesicle (arrow) is forming within the lamellipodium directly under a glial process which contains several ribosomes. Based upon an analysis of serial sections this glial structure was club-shaped. Calibration bar is 1 µm.

As early as E28 a majority of axons contained neurofilaments (Fig. 7B). The mean number of neurofilaments per axon at this age was about 6, but the range was large—from 0 to 50. The density of neurofilaments in axons at this early stage of development surprised us because both Peters and Vaughn (1967) and Pachter and Liem (1984) have reported that optic axons of rats essentially lack neurofilaments until about a week after birth—an age roughly equivalent to E-50 in the cat. Neurofilaments may be labile during fixation early in development because the concentration of the heavy neurofilament subunit is so low (Willard, 1983; Pachter and Liem, 1984). In adult mammals, neurofilament polypeptides and microtubules are transported together at a rate of about 0.25 mm/day (Black and Lasek, 1980). Since ganglion cell axons grow at rates of 1 to 2 mm/day (Rager, 1980; Halfter and Deiss, 1984) and possibly in spurts of up to 3 or 4 mm/day (compare with Schreyer and Jones, 1982), and since neurofilaments and microtubules are prominent in growth cones as early as E23 (see below), it is reasonable to conclude that the transport of these two cytoskeletal constituents is considerably faster early in development than at maturity (also see: Droz, 1963; Grafstein and Murray, 1969; Hendrickson and Cowan, 1971).

Figure 13

Fig. 13. Distribution of organelles in axons and a lamellipodium at E33. The lamellipodia (asterisk) contains little other than microfilaments. As is generally true of osmicated tissue, these fibrils are not clearly organized. Microtubules within axons have a diameter of about 15 nm and the neurofilaments of 7–8 nm. Nearly all axons contain cross-sections through smooth endoplasmic reticulum, lending support to the idea that the reticulum is a nearly continuous system. Glial process (star) contains numerous ribosomes. In the center of the field is a large shaft of a growth cone that contains about 40 microtubules, a single mitochondrion, and smooth endoplasmic reticulum. Small unspecialized contact points between the lamellipodium, adjacent glia, and the large growth cone core are marked by arrowheads. Calibration bar is 1 µm.

Axon ultrastructure did not appear to change qualitatively during the later stages of prenatal development. However, we found that after E53 it was at times difficult to distinguish axons from small glial processes. Many small glial processes did not contain ribosomes and at first inspection looked much like axons. Generally, however, glial processes were less circular than axons in cross-section, were more frequently cut at oblique angles, contained a higher concentration of intermediate filaments than axons did of neurofilaments, and usually contained less than 3 microtubules (Fig. 24B). Axons typically contained a minimum of 5 microtubules. The distinction between axons and glial processes was thus based upon a constellation of properties—ultrastructure, form, and position. Only 5% of all processes presented a classification problem, and therefore, we do not think that the accuracy of the counts was significantly degraded either by the inclusion of glia or the exclusion of axons.

 

Ultrastructure of growth cones

Growth cones of retinal ganglion cells were large, often had complex shapes, and possessed an organellar composition that allowed them to be easily distinguished from axons and glial processes (Figs. 7A, 813, 21). Growth cones have two distinct parts: a distal fringe and a bulbous central core. The ultrastructure and shapes of these segments differed radically, and as a consequence, in single transverse sections through the optic nerve growth cones displayed a range of characteristics that differed depending on how far from their tips they had been sectioned (Fig. 10).

The most distal parts of growth cones were almost entirely made up of sheet-like extrusions of membrane called lamellipodia. These had simple ultrastructure and contained merely a mesh of microfilaments and occasional clear and dense-core vesicles (Figs. 12, 16). Although microtubules, neurofilaments, and mitochondria were common within the core region of the growth cone (Figs. 7A, 10, 11), these organelles were almost entirely absent from lamellipodia. Lamellipodia were apposed either to the surfaces of glial cells, other growth cones, or axons. In single cross-sections, the largest lamellipodia had a breadth of about 5 µm (Fig. 8), a thickness of 0.1–0.3 µm, and judged from growth cones sectioned longitudinally, they were up to 25 µm long (Fig. 10). The number of fibers growth cones were apposed to was in some instances remarkably high—up to 74 in single transverse sections. However, even growth cones in very small fascicles occasionally had very elaborate lamellipodia, and it therefore does not appear that the formation of lamellipodia is strictly related to the number of potential neighbors.

Filopodia, small finger-like protrusions extending out from the growth cones, were surprisingly rare at all stages of development. At first sight it may seem that most of the processes extending from growth cones are small radial spikes (e.g., Fig. 8). However, in single longitudinal sections and in short series of transverse sections it quickly becomes apparent that these processes are virtually without exception sheets of membrane. If there are any filopodia, then in transverse sections they should appear as small oval profiles, about 0.1 to 0.3 µm across their short axis, that contain microfilaments but no microtubules or neurofilaments (see for example, the abundance of such profiles in figure 11 of Bastiani et al., 1984). In a sample of micrographs of the E28 nerve that contained 20,000 axons and about 400 growth cones, only 15 such presumed filopodial profiles were found (Fig. 15).

The core region of the growth cones from which the lamellipodia stem usually had a caliber nearly 3 times greater than was typical for axons (Figs. 7, 8, 11). This core region usually contained a great variety of organelles, including 40–60 nm clear vesicles, coated vesicles, coated pits, dense-core vesicles, mitochondria, microfilaments, a substantial amount of smooth endoplasmic reticulum, microtubules, and neurofilaments. All of these organelles were also found in axons, although in lesser numbers and in differing concentrations. For instance, the core region of growth cones often contained in the neighborhood of 15 microtubules and occasionally twice this number were noted in single sections. In comparison, the mean number of microtubules in typical axons with diameters ranging from 0.3 to 0.4 µm, was 5–6. The only structure seen exclusively in growth cones was a maze of tubules resembling smooth endoplasmic reticulum that was usually enmeshed in a nest of neurofilaments (Fig. 11). The cisternea were more darkly stained than is typical for perinuclear endoplasmic reticulum.

Although nearly all growth cones had certain features in common, there were nonetheless several notable qualitative differences in their form and ultrastructure. In large part, this was due to sectioning growth cones at different distances from their tips. However, some differences appeared to be age-related. At E19 and E23, growth cones in the optic stalk had only a small number of stubby protrusions, which did not seem to merit either the term lamellipodia or filopodia (Fig. 6A, B, C). With a few exceptions, the form of these first growth cones appeared to be particularly simple. Furthermore, the concentration of cytoskeletal components, particularly microfilaments appeared substantially less in this first group of growth cones than in those at E28 and E33.

Bastiani and Goodman (1984) have shown that in embryonic grasshoppers, filopodia of certain growth cones selectively penetrate into the core of other growth cones and induce the formation of coated pits. They have suggested that coated pits and vesicles mediate fiber-fiber recognition and perhaps ultimately the direction of axonal growth. Given this result, and the earlier work of Altman (1971) and Vaughn and Sims (1974) in which coated vesicles had been linked with early stages of synaptogenesis, we decided to examine the distribution of coated pits in a large sample of fibers at E28. We found that 9 of 410 growth cones (2.2%) had prominent coated pits with diameters of 50-90 nm on their surfaces (Fig. 14). In addition, similar coated pits were also found on the surfaces of 30 (0.14%) out of 20,400 axons (Fig. 14). After correcting for the 4–5-fold greater perimeters of growth cones, we conclude that coated pits are about 4 times more common on growth cones than on axons. The direction of movement of these coated pits and vesicles is not known. However, the long necks connecting the main body of the coated pit to the surface suggests strongly that at least some are pulling away from the plasma membrane.

Figure 14
 

Fig. 14. Cytoplasmic organelles in axons and growth cones at E28. Coated pits, marked by arrowheads, are shown forming on both a growth cone and two axons. The upper arrow points to a dense-core vesicle; the lower arrow to a multivesicular body. Very small arrowheads mark junctions between adjacent glia (gl). Asterisks mark growth cones and their lamellipodia. A single filopodial profile is circled in white. Download a high-resolution 1.2 MB image to resolve these fine ultrastructural details.

Although we found no evidence that neighboring growth cones ever had processes that protruded into one another as in grasshopper (Bastiani and Goodman, 1984), we did note two cases in which glial cell processes indented or deeply penetrated the surface of growth cone lamellipodia. In both cases, one or two 50–nm vesicles were found fused with the plasmalemma of the growth cone at these sites of intimate contact (arrow in Fig. 14) suggesting that some inductive event, perhaps similar to that described by Bastiani and Goodman (1984) between growth cones in the grasshopper embryo, is taking place. Examination of serial sections revealed that the glial processes were spines rather than ridges.

One of the most clear-cut distinctions in our material between axonal growth cones and pseudopodia of glial cells was the complete absence of ribosomes in growth cones and the very high density of ribosomes in glial processes (Figs. 6–8). In this respect our results agree with those of Pfenninger and Bunge (1974) and Williams and Rakic (1984). Another distinction was that mitochondria within the cores of growth cones were usually considerably smaller than those in glioblasts (compare mitochondria in Figure 7A, B). A final criterion, especially useful at later stages of development (ca. E39, see Figure 21), was the density of intermediate filaments: Density was high in glial processes and was much lower in axonal growth cones.

Figure 15

Fig. 15. Distribution of growth cones at E33. Sixty-two micrographs, each covering 90 µm2 of the nerve, were scored for growth cones. Corrections were made for the proportion of each micrograph containing glial processes and all calculations excluded regions occupied by glial cells and their processes (roughly 25% of the nerve at this age). The growth cone density per 100 µm2 of nerve was split into four ranges; a high range (>16 growth cones/100 µm2) is represented by the largest spots (the average for this group was 22 per 100 µm2 and the highest value was 28 per 100 µm2). Spots that actually interrupt the outline of the edge of the nerve represent micrographs taken at the extreme periphery. The smallest spots represent regions containing fewer than 6 growth cones/100 µmm2.


 

Number of growth cones. At E28, 2.0% of all fibers were growth cones. They had perimeters between 3 and 6 µm with a mean of 4 µm—about 4 times that of axons. By E33 the percentage of growth cones had dropped to approximately 1.1%. Their perimeters were on average slightly larger (about 15%) than those at E28, and although the percentage of growth cones in the nerve was higher at E28 than at E33, the total number of growth cones actually peaked at E33; nearly 3,000 were distributed in a non-uniform fashion (see below) throughout the nerve. Growth cones often have bifurcating lamellipodia in the optic nerve of embryonic primates (Williams and Rakic, 1984), and these percentages probably overestimate the total number of growth cones. An additional 4–10% of all axons were unusually large (>0.6 µm) at these ages (Fig. 2A, 7A, 8). Since these large axons disappeared with the same time-course as did growth cones they almost certainly were cross-sections through the long flared shanks of growth cones. By E39 merely 0.2–0.4% of all fibers were classified as growth cones (Table 1), and these appeared to be as large and complex as growth cones at E28 and E33. For example, the remarkable growth cone reproduced in 21A had a span of 10.2 µm, a perimeter of approximately 40 µm, and made contact with 42 axonal neighbors in a single transverse section. By E44 there were essentially no growth cones in the nerve. One of the E44 nerves was completely devoid of growth cones, whereas the other contained a total of less than 20 growth cones.

Figure 16

Fig. 16. The optic nerve at E28; montage of low-power electron micrographs. At E28 midorbital sections of the optic nerve have an elliptical shape with axes approximately 100 and 150 µm long. In transverse section the processes of glial precursors divide the nerve into clearly delimited fascicles of axons (Figs. 7, 16). At this level the nerve is made up of 105 fascicles that collectively contain approximately 42,000 axons and 800 growth cones. Peripheral fascicles are only slightly smaller on average than those at the center but contain fewer fibers and about twice as many growth cones. Growth cones are scattered widely. Remarkably few glial processes intruded within the fascicles themselves. The figure is reproduced at about one-half the magnification as the E19 optic stalk (Fig. 3). The remarkable transformation in nerve architecture is evident. Neuroepithelial cells are no longer present, a distinct glial limiting membrane insulates the fibers, glial cells are dispersed throughout the nerve, and no remnant of the lumen is visible. At this age the nerve is avascular. Calibration bar is 10 µm.

Distribution of growth cones. There was a moderate central-to-peripheral difference in the proportion of growth cones at E28 and E33 (Fig. 15). At E28 there were twice as many growth cones at the nerve’s periphery, within 12 µm of the margin, as there were at the center—10 versus 5 per 100 µm2. By E33, the absolute proportion of growth cones had decreased and the difference between center and periphery was only slightly more pronounced: densities remained about 5.0 per 100 µm2 at sites located farther than 10-15 µm from the edge (7.2 per 100 µm2 if glial processes are excluded) , but were 3 to 4-fold greater at the edge (Fig. 15). Growth cone density reached a plateau within 10 or 20 µm of the edge of the nerve, and consequently within central and pericentral parts of the nerve the density of growth cones showed little or no systematic change (Fig. 15); virtually all central fascicles contained several growth cones.

Figure 17

Figs. 17. Dying fibers in the optic nerve at E28. (A) A peripheral fascicle that contains dying axons (arrowheads). Their axoplasm is dark and membranes appear to be disintegrating. The ultrastructure of the majority of axons and growth cones, however, is normal.(B) Necrotic axon marked in A at higher magnification. (C) A disintegrating axon marked in A. Calibration bars 1 µm; B and C at same magnification. Download a high-resolution 600 KB image.

At a local, fascicular level of analysis there often appeared to be a slight central-to-peripheral gradient. Growth cones were somewhat more frequently located at the outer edges of fascicles, positioned between other fibers and glial processes (Fig. 7A). However, there were certainly numerous exceptions; many growth cones were found buried among axons in the center of large fascicles without evident glial contact (Figs. 7B).

At E39 there was still a clear spatial gradient in growth cone distribution. Between 70 and 80% of the growth cones were located in a 50-µm-wide annulus of the nerve that covered half the cross-sectional area. The remaining 20–30% were situated more than 50 µm from the nerve’s edge (Figs. 22B). In contrast to earlier ages, at E39 substantial parts of cross-sections of the nerve were almost completely devoid of ingrowing fibers. This was true not only of central regions, but also of fairly large peripheral sectors. The overall central-to-peripheral gradient of growth cone distribution may reflect the rough central-to-peripheral gradient in the generation of ganglion cells across the retinal surface (Walsh et al., 1983), or as suggested by Bohn et al. (1982) and Silver and Rutishauser (1984), this may simply reflect a tendency for growth cones to grow close to the outer margins of the optic nerve.

Figure 18

Fig. 18. Necrotic axons. A and B: E33; C and D: E39; E and F: E53; G and H: E61. Axons were counted as necrotic if they contained dark and mottled axoplasm. Most axons, such as those in B, C and H, are partly disintegrated whereas others contain large accumulations of dense material (A, B, D). Other axons (i.e., G) are partly intact and the dense inclusions may either be necrotic mitochondria (a characteristic of early stages of degeneration) or may be debris these axons have phagocytized. Calibration bar is 1 µm for A through H. Download a high-resolution 425 KB image.


 

 

Fiber necrosis

Early axon necrosis. As early as E28, fibers that contained dark inclusions, dense lamellar structures, dilated and very electron-dense mitochondria, large vacuoles, and disrupted plasma membranes were regularly encountered in the nerve (Figs. 17, 18A, B). Such a constellation of features are associated with acute stages of axonal disintegration during normal development in many parts of the nervous system (see Discussion). Some of the necrotic axons simply contained large electron-dense autolytic inclusions (Fig. 18A), that in many cases may have been necrotic mitochondria or lysosomes. Others showed more severe signs of degradation: the entire axoplasm was dark and mottled, microtubules and neurofilaments could not be resolved, and the axolemma was ruptured (Fig. 17B, C). The size of degenerating axons was variable, but in general they were about twice as large as their neighbors.

Figure 19

Fig. 19. Usually and possibly necrotic growth cones at E33. (A) Large growth cone with an extraordinarily high vesicular content. X 25,000. (B) The large inclusion and the accumulation of intermediate filaments in this neurite suggest that this process may be starting to die.(C) Dilated growth cone (asterisk) with hyperplasia of neurofilaments and an accumulation of large dark-rimmed vesicles. (D) Large fiber at an early stage of necrosis. The accumulation of neurofilaments in degenerating fibers is usually transitory and is followed by the condensation of the cytoplasm (cf., Lund, 1978, p. 45). All calibration bars are 1 µm. Download a high-resolution 600 KB image.


 

The percentage of necrotic axons was high at E28—about 0.23%. At E33 and E36 only about 0.10% were obviously necrotic. In none of these nerves was there any strong evidence of regional differences in the proportion of dying axons—central, intermediate, and peripheral parts of the nerve contained roughly the same percentage (Table 2).

There were also a number of axons that were abnormal in some ways, but not so strikingly abnormal as to enable us to confidently categorize them as necrotic. Such ambiguous fibers were not included in counts of necrotic axons. They may represent early stages of necrosis or they may have been axons that had taken up the debris of other neighboring necrotic axons. Their numbers varied roughly in proportion to the number of axons that were unambiguously necrotic.

Figure 20

Fig. 20. Large necrotic fibers at E33. (A) An early stage of organelle accumulation and lysosome formation. (B) Large fiber with condensed, vesicular cytoplasm. Note neighboring growth cone (asterisk) above necrotic fiber. (C) Ruptured fiber. (D) Highly condensed axoplasm that is either enveloped by or actually within a large and apparently normal fiber. Both calibration bars are 1 µm; B, C, and D at same magnification. Download a high-resolution 374 KB image.


 

There were many necrotic axons in the nerve several days before retinal fibers penetrate the dorsal lateral geniculate nucleus (ca. E32, Shatz, 1983). This raised the intriguing possibility that some fibers die while still extending through the nerve. We therefore decided to search for necrotic axon terminal bulbs (Lampert, 1967)—the equivalent of necrotic growth cones (Yamada et al., 1971). The entire cross-sections of the E28 and E33 nerves were scanned at high magnification (X20,000-30,000). Only 5 large necrotic fibers, that may have been the swollen ends of degenerating axons, were found at E28, but at E33 nearly 20 extraordinarily large necrotic fibers and highly atypical growth cones were found (Figs. 19, 20). In every case these structures were clearly not of glial origin: they never contained ribosomes, rough endoplasmic reticulum, or the large mitochondria characteristic of glial cells. Several of these necrotic fibers had features intermediate between growth cones and necrotic axons (Fig. 19). Some were extraordinarily large, even in comparison with growth cones, and contained regions almost exclusively occupied by bundles of neurofilaments (Fig. 19B, C, D). These fibers appeared essentially identical to previous descriptions of reactive and dystrophic axon terminal bulbs described in the adult nervous system (e.g. Lampert, 1967).

Figure 21

Fig. 21. (A) One of the largest and last growth cones encountered in this study at E39. This growth cone, located within 2 µm of the edge of the nerve, has a perimeter of about 25 µm and has 42 neighbors in this section. The density of all cytoplasmic components, even microfilaments, is low. Several types of vesicles, including a single dark-core vesicle, are present in the growth cone. (B) A growth cone from the central fascicle. The ultrastructure of central and peripheral growth cones did not differ significantly, and although there is a marked difference in size between these two growth cones, this difference could easily have resulted from the level of section. Both calibration bars are 1 µm. Download a high-resolution 655 KB image.


 

The period of heavy axon loss. An average of about 0.3% of axons in the optic nerve were necrotic between E39 and E48. The characteristics of necrotic axons during this period (Fig. 18E, F) did not differ in any respect from those described in the previous section at earlier stages. There did not appear to be any consistent spatial gradients in the location of necrotic axons. They were scattered throughout the nerve. By E53 their incidence was quite low—less than 0.05%, and similar low values were found as late as P2.

Figure 22

Fig. 22. E44 optic nerve close to the periphery. The fibers in this fascicle appear to fall into two size groupings. Large axons in the upper half may either represent newly ingrown fibers that are sectioned close to their expanded tips or may simply represent a class of large axons. Calibration bar is 1 µm. Download a high-resolution 434 KB image.


 

In order to calculate the total production of axons it was necessary to estimate the number of axons lost early in development while other additional fibers were still extending through the nerve. We estimated this number by first determining the time required to clear away the debris of dying axons during the period when all changes in the total fiber number could be attributed to fiber loss; that is, after all growth cones had grown through the nerve. Over a 216 hour period between E39 and E48 approximately 375,000 axons were eliminated. Thus an average of 1,700 axons were lost per hour during a period when the number of necrotic axons was about 0.3% of the fiber population. Naturally however, more fibers were actually eliminated per hour at E39 and E44 than at E48. It was therefore necessary to correct for the severity of axon loss at each age bearing in mind the total number of axons in the nerve. The ratio of the number of necrotic axons in single cross-section through the nerve to the number lost per hour gives an estimate of the time required to clear away axonal debris of about 1 hour. For comparison, the clearing time of the cell bodies of retinal ganglion cells has been estimated to be 2 to 4 hours in neonatal hamster (Sengelaub and Finlay, 1982) and about 3 hours in the spinal cord of Xenopus tadpole (Hughes, 1961). Using our 1-hour approximation, we estimate that between E28 and E39 from 0.09% to 0.26% of the fiber population was eliminated every hour (see Table 1). Subtracting the daily loss while correcting for differences in the incidence of loss at each age indicated that about 150,000 axons were lost between E28 and E39. We stress that this number is only a rough approximate because the number of individuals on which the calculation is based is low and because it is possible that clearing time varies as a function of age (see below) or even time of day (see Vogel, 1978).

Figure 23

Fig. 23. E53 optic nerve. Fascicles are subdivided repeatedly by astrocytic processes. Axons have much paler cytoplasm and far fewer intermediate filaments than do astrocytic fibers and astrocytic growth processes. Calibration bar is 1 µm. Download a high-resolution 536 KB image.


 

Late stage of axon loss. There were very few necrotic fibers in the optic nerve during the perinatal period. In fact, in the group of micrographs used to estimate total axon number at E61 there were only 2 unmyelinated necrotic fiber among about 7,700 that were counted. Thus, the percentage of necrotic fibers is under 0.05%. However, at E61 there were several necrotic glial cells (Fig. 24A). This wave of glial necrosis is probably related to the proliferation of oligodendrocytes, and it is tantalizing to speculate that even glia are overproduced during development (cf. Mori and LeBlond, 1970; Hildebrand, 1971). Three of these necrotic glia were found within a sample of the nerve that covered merely 3.5% of its area. These observations are remarkably similar to those of Chu-Wang and Oppenheim (1978b), who previously described the necrosis of Schwann cells during the myelination of the ventral roots of the chick embryo.

Figure 24

Fig. 24. Myelination at E61. In (A) 3 axons marked by asterisks are in the process of being enveloped by glial tongues. Necrotic glial cells (dark, mottled region) and processes were common at this age and their presence may be related to the genesis of oligodendrocyte or the death of precursor cells. (B) Axons and astrocytic processes at E61 are hard often to distinguish from one another. Astrocyte fibers, a few of which are labeled (gl), generally have many intermediate filaments and less than 3 to 4 microtubules. Multivesicular body (small arrow) and axo-axonal invagination (large arrow). Calibration bars are 1 µm. Download a high-resolution 510 KB image.


 

By postnatal day 12, in agreement with Moore et al. (1976), about 25% of the fibers were myelinated. These fibers were generally considerably larger than non-myelinated or pro-myelinated axons (Fig. 25A, B). It was of interest to determine whether as suggested by Rager (1980) and Sefton and Lam (1984), necrosis was limited to smaller unmyelinated axons. In a sample of 15,000 axons, about 25 necrotic myelinated axons and 3 necrotic unmyelinated axons were found. The necrotic myelinated fibers had extremely dense axoplasm often full of intermediate filaments (Fig. 25C). In comparison to the perinatal cats (E61, and P2), the incidence of necrotic axons at P12 was high. This observation makes sense because the clearing time for a myelinated axon is much longer than that of a naked axon—on the order of weeks and months (Nathaniel and Peese, 1963; van Crevel and Verhaart, 1963; Cook and Wisniewski, 1973), and this may explain the fact that necrotic axons were seen in the nerve of the older kittens (P36 and P84), even though the population of fibers had reached adult levels. We are not the first to demonstrate dying myelinated axons in the kitten’s optic nerve: Cook et al. (1974) reported that 3-5% of myelinated axons at P7 showed degenerative changes but that by P-35 less than 1% showed similar evidence of necrosis. We counted far fewer necrotic axons in our P12 and P36 nerves. Possibly our criteria were more stringent. In any case, it is evident that both unmyelinated and myelinated axons undergo degeneration during normal development. This is also the case in the ciliary nerve (Landmesser and Pilar, 1976), the trochlear nerve (Sohal and Weidman, 1978), and the ventral roots (Chu-Wang, and Oppenheim, 1978b). Clearly, the hypothesis advanced by Sefton and Lam (1984, p. 115) “that axons lost during periods of cell death in any system will prove to be unmyelinated” is not tenable.

Figure 25

Fig. 25. Optic nerve at P12, several days after eye opening. (A) High-power light micrograph of the nerve during myelination. Large numbers of oligodendrocytes are intermixed with axons and as a consequence fascicular organization is disrupted. (B) Fine structure of the nerve during myelination. The majority of axons in this field are either myelinated or are being enwrapped by glial processes. There is an increase in the amount of extracellular space during myelination. (C, D) Necrotic myelinated axons (see text).Calibration bar is 10 µm in A and otherwise, 1 µm. Download a high-resolution 476 KB image.


 

 

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DISCUSSION

Synopsis. We have shown that a total of 800,000 to 900,000 axons grow into the optic nerve of the cat between E19 and E39. At the peak of axon ingrowth (E28 to E33) several thousand growth cones are distributed throughout the nerve, preferentially around the periphery but also within its core. Surprisingly, during this period of axon ingrowth numerous necrotic fibers are found throughout the nerve. However, the number of necrotic fibers does not peak until about E44. A total of 80% of all axons are lost over a protracted period that begins about the same time that fibers reach their targets (E28), extends through birth (E65), and lasts until several weeks after eye opening.

Growth cones in the optic nerve

Identification of growth cones. The growth cones we have studied in the cat’s optic nerve are similar in shape and ultrastructure to large velamentous axonal growth cones first described in detail by Skoff and Hamburger (1974). These authors analyzed serial sections of embryonic chick spinal cord and provided a thorough set of criteria by which to distinguish axonal growth cones from dendritic growth cones and growing neuroepithelial or glial processes. We have relied upon these criteria extensively. Our description is also generally in agreement with several recent electron microscopic studies in which retinal ganglion cell growth cones were reconstructed (Bodick, 1980; Easter et al., 1984; Williams and Rakic, 1984, 1985a). Perhaps the only notable difference is that Bodick (1980) reported numerous filopodia—he called them microspikes—extending out from growth cones of embryonic zebrafish, whereas we saw very little evidence of filopodia in the cat’s optic nerve at any stage of development. Filopodia have not been found in extensive three-dimensional reconstructions of growth cones in the optic nerve of primates (Williams and Rakic, 1984, 1985a).

Ultrastructure of growth cones. The axonal growth cone can be divided into two segments: a distal fringe, in our material made up almost exclusively of lamellipodia; and a core. This distinction is by no means original to our work; it can be traced back to early light microscopic studies of growth cones (Harrison, ’10; Speidel, 1933; Pomerant, 1967), and has been described in detail in the in vitro ultrastructural studies of Yamada et al. (1970, 1971) and Bunge (1973). In examination of several thousand growth cones in situ in this study it was clear from the outset that, as reported first reported by Tennyson (1970) and Yamada et al. (1970), the distal fringe of the growth cone contains little more than a mesh of microfilaments that are closely associated with the inner wall of the plasma membrane and scattered clear vesicles. In contrast, the more proximal core, that Williams and Rakic (1984) have shown is located about 12 µm behind the leading edge, contains the cytoplasmic machinery of the growth cone. This is the site at which microtubules and neurofilaments terminate, and as a consequence it is also the site at which normal axoplasmic transport ends (reviewed in Lasek, 1982). Here the axoplasm contains several classes of vesicles, mitochondria, and a meshwork of what may be smooth endoplasmic reticulum (e.g., Figs. 10, 11).

As in the lamellipodia, large accumulations of clear, 40 to 120-nm vesicles were only rarely encountered in the core. Their scarcity surprised us because several previous studies have emphasized that aggregates of vesicles are a key feature of growth cones and that they are probably required to sustain rapid axon elongation (Bodian, 1966a; Del Cerro and Snider, 1968; Kawana et al. 1971; Del Cerro, 1974; Pfenninger and Bunge, 1974). However, a large complement of vesicles is not actually needed to sustain axon growth. We have shown that axons invade the optic stalk as early as E19. These first fibers reach the dorsal lateral geniculate nucleus and the superior colliculus before E28 (Shatz and Kliot, 1982; Shatz, 1983). Since the distance between the posterior part of the eye and target nuclei is under 1 cm, this initial axon contingent grows into the brain at an average velocity of between 0.5 and 1 mm per day. Therefore only enough membrane must be added at the surface to increase the length of a narrow-caliber axon by an amount equivalent to the net forward movement of the growth cone. Even at a velocity of growth of 2 mm/day this amounts to only 2000 µm2 of membrane for a typical axon with a diameter of 0.3 µm. The fusion of three 50-nm vesicles per second with the plasmalemma is sufficient to account for this growth. A large membrane reservoir is therefore probably not essential, and those accumulations of vesicles that are occasionally encountered (e.g., Fig. 8) may be related to the retraction and internalization of lamellipodia, or may even be artifacts of glutaraldehyde fixation (Hasty and Hay, 1978; Nuttall and Wessells, 1979; Falls and Gobel, 1979).

The shape of growth cones. There were clear age-related differences in the form and fine structure of growth cones. The differentiation between the distal fringe and the core was only rudimentary in the first growth cones that extended through the ducts of the optic stalk (Fig. 6); lamellipodia, when seen at all, were short and thick; microfilaments and neurofilaments were sparse. In contrast, growth cones that later grow within dense fascicles of axons in the optic nerve of older fetuses (E28 and up) were, with few exceptions, characterized by large lamellipodia and organelle-rich core regions. This difference may have several causes. First, the simple shapes of the earliest growth cones may reflect the simple tubular environment of the optic stalk. Second, the comparatively low concentration of cytoskeletal components in these young growth cones may not be sufficient to support large lamellipodia. Third, the shape of growth cones may be influenced by the molecular composition of the surfaces along which they grow and by their velocity of growth, and both of these factors probably changes with age (Agiro et al., 1984). Finally, it is possible that all growth cones, irrespective of developmental stage, initially have simple morphology and that the pertinent distinction may actually be the age of individual growth cones—not that of the animal. Because ganglion cell generation proceeds from a region near the macula outward toward the retinal margin (Mall, 1893; Mann, 1964; Walsh and Polley, 1985), growth cones in nerves of older animals are almost invariably located at greater distances from the cell body than those in nerves of younger animals.

Distribution of growth cones. The tendency of fibers to add to the outer margins of the optic stalk and optic nerve was established in the last century by Müller (1874), Assheton (1892), and Robinson (1896). Assheton went so far as to state that fibers actually grew outside the stalk, like vines along a tree trunk. Robinson recognized correctly that fibers pushed through spaces between the distal ends of neuroepithelial cells and were at all points actually within the central nervous system. In more recent studies, it has been emphasized that growth cones of optic axons in a variety of species normally grow against the endfeet of neuroepithelial cells and near the extreme outer margins of the optic nerve (Silver and Sapiro, 1981; Rager, 1983; Silver and Rutishauser, 1984). Although our results substantiate and amplify some of these observations, we stress that the central-to-peripheral gradient in growth cone distribution in the cat is actually quite modest at early stages of development. Growth cones are found in all fascicles, including those close to the center of the nerve. Even as late as E39, when the gradient in the distribution of growth cones is comparatively steep, growth cones are still not uncommon in central parts of the optic nerve. Essentially the same pattern of fiber addition is also found in the optic nerve, chiasm, and tract of rhesus macaque fetuses (Williams and Rakic, 1985b). In this species, growth cones are initially widely distributed in all parts of the pathway, but at later stages of development growth cones become progressively more restricted in their distribution and are eventually found only within 100 µm of the glial limiting membrane.

Necrotic fibers

Necrotic fibers occur commonly in central fiber tracts, peripheral nerves, and the gray matter at early stages of development (Bodian, 1966b; Berger, 1971a; Das and Hine, 1972; Reier and Hughes,1972; Pannese, 1976; Landmeser and Pilar, 1976; Chu-Wang and Oppenheim, 1978b; Cunningham et al., 1982). There is substantial agreement that young unmyelinated axons undergoing degeneration contain focal accumulations of autolytic debris, large vacuoles, and dense lamellar inclusions, some of which are probably disintegrating mitochondria. As might be expected, these spontaneously degenerating axons have ultrastructural features often indistinguishable from unmyelinated axons in the mature nervous system undergoing experimentally-induced Wallerian degeneration (e.g., Brooke et al., 1965; Lampert, 1967; Roth and Richardson, 1969; Berger, 1971b; Dyck and Hopkins, 1972; Reier and Webster,1974; Bohn et al., 1982; Williams et al., 1985). Axons that we classified as necrotic in the optic nerve fit this description well (Figs. 17, 18). Degenerating axons in the nerve were surrounded by normal fibers and it is therefore not likely that they were artifacts of fixation or tissue processing. Furthermore, their incidence was greatest in the period during which the axon population was decreasing rapidly, and conversely very few such profiles were seen when the population was relatively stable.

The only previous study in which an attempt has been made to quantify the incidence of axon necrosis as a function of age is that of Landmesser and Pilar (1976). They demonstrated that during the peak of axon loss from the ciliary nerve of the chick embryo nearly 7–8% of all fibers were necrotic. Based upon their data it is possible to calculate a clearing time of axons of about 6–7 hours for axons. In contrast, we have estimated that it takes merely 1 hour to clear away the debris of an unmyelinated axon at a given level along the optic nerve. Two assumptions underlie our estimate: First, that all axon loss is due to necrosis. If, however, a significant percentage of axons are retracted without autolysis of their constituents or disruption of membranes, then the clearing time of 1 hour will underestimate the true clearing time. Second, we have assumed that the duration of degradation of small unmyelinated axons does not vary significantly with the time of day or fetal age. At present we have no reason to suspect that these are relevant variables, but if this proves incorrect then our estimate of clearing time will require adjustment. Furthermore, it is important to point out that our estimate of clearing time does not allow us to calculate the time required to eliminate the entire axon. If the process of axon elimination is analogous to burning a fuse, then we have simply determined the time required to burn a short piece of the fuse. The entire process of necrosis probably takes considerably longer than an hour. In fact, Rager (1980, p. 81) has estimated that the time constant for the degeneration of retinal ganglion cell axon in the chick is nearly 4 days.

It is unclear what removes axonal debris from the nerve. In other systems in which there is axon loss, glial cells or marcrophages have been implicated in the phagocytosis of axonal debris. In the ventral roots, for instance, the rapid disintegration of more than half the axon population during embryogenesis is associated with the uptake of debris into Schwann cell phagosomes (Chu-Wang and Oppenheim, 1978b) and similarly, during the early stages of Wallerian degeneration of the optic nerve, reactive astrocytes engulf large amounts of axon and myelin debris (Reier and Webster, 1974). But we have been unable to find convincing signs of a similar process in the optic nerve during normal development, and in several instances, it appeared that other optic fibers rather than astrocytes or macrophages had phagocytozed axon debris (Fig. 18G, Fig. 20D).

A number of very large necrotic fibers were noted in the optic nerve at the peak of axon ingrowth (Figs. 19, 20). None of these contained ribosomes, and because no similar processes were found in continuity with cell bodies we are confident that we have not misidentified the processes of glial cells. The large size of these necrotic fibers and their occurrence within fascicles of otherwise normal axons suggests that they may be the tips of dying fibers—the swollen terminal bulb described by Lampert (1967). Since these were not observed beyond E39, after all growth cones had grown through the nerve, it is improbable that they were simply sections through dilated regions of normal-caliber axons in which debris had accumulated. We think it is likely that such structures as shown in Figures 19 and 20 are growth cones in the early stages of degeneration. As described by Speidel (1933), the first indications of impending death of a growing axon in the tadpole is the retraction of its lamellipodia and filopodia (he called them sprouts) and the balling-up of the growth cone. Similarly, the retraction of growth cones in vitro is often associated with large accumulations of neurofilaments in discrete regions from which all other organelles are excluded (Yamada et al. 1970, 1971). This is precisely one of the most striking features of the growth cones reproduced in Figure 19. Such hyperplasia of neurofilaments is also seen commonly in terminals of retinal axons undergoing Wallerian degeneration (Guillery, 1970; Lund, 1978).

Magnitude of fiber loss and the estimate of total axon production

We have divided the loss of fibers in the cat’s optic nerve into three phases. The first phase begins early in development, before axons have arborized extensively within their central targets. The number of axons lost during this period is not great—probably under 50,000. The second, more rapid phase of axon loss begins a few days before the peak fiber population is reached at a stage when the last growth cones are growing through the nerve and while target nuclei are being innervated. During this period, which lasts from about E-36 to E53, as many as 500,000 axons are lost. The third phase of axon loss starts at about E53 and extends until about P36. During this long-lasting phase the rate of loss is very low, but even so, approximately 100,000 axons are eliminated. Although we can divide axon loss into three periods, we currently know disappointingly little about the causes that underlie the loss, and whether any given period is associated with a single dominant process.

Because the period of axon addition to the fetal cat’s optic nerve overlaps that of axon loss for at least 11 days, many axons are eliminated even before the peak population is reached. As a consequence, to calculate the total production of axons it is necessary to determine how many fibers are lost before the peak and how many fibers are added after the peak. Although the need to provide for such a correction was recognized in at least one previous quantitative study of the developing optic nerve (Rakic and Riley, 1983a), our work is the first in which an attempt has been made to provide the correction. On the basis of the time it takes to clear away axonal debris (approximately 1 hour), we estimated that 150,000 axons are lost before the peak is reached. In contrast, very few axons—probably fewer than 10,000 or 20,000—are added after the peak. Therefore, a total of about 800,000 to 900,000 axons are produced and grow into the optic nerve—5 to 6 times the number in the adult nerve (Williams et al., 1983; Williams and Chalupa, 1983b; Chalupa et al., 1984; Williams et al., 1985).

Overlapping periods of axon generation and loss are common in many different parts of the nervous system. One of the most remarkable cases involves the development of the electric lobe (a cranial nerve nucleus) of the electric eel, in which neuron and axon production is vigorous during two periods of cell death (Fox and Richardson, 1982, 1984). Similarly, the overlap in the proliferation and death of neurons is so extensive in the spinal cord of Xenopus (Hughes, 1961; Prestige, 1965) and in the dorsal root ganglia of chick (Carr and Simpson, 1982) that the total production of neurons is nearly twice as great as the peak population.

The time course and the magnitude of the developmental fluctuations in the fiber population of the cat that we have described in this paper differ in detail from the results of Ng and Stone (1982). They reported that there were 450,000 to 483,000 axons in the optic nerve from E-42 until as late as E-55. In contrast, we have shown that a peak of about 700,000 is reached by E39 and that the fiber number decreases to the range of 200,000-300,000 as early as E52. The discrepancies between their findings and the results reported here could conceivably reflect strain variation between cats. However, we think it is more likely that the method Ng and Stone used to estimate gestational age accounts for these differences. In their study several fetuses were removed by cesarean section from a litter of unknown age. The gestational age of these fetuses was then estimated from the date that the remainder of the litter was born, on the assumption that gestation was 65 days long. However, gestational age at birth is normally quite variable in the domestic cat (see Methods) and this variability is exacerbated by subjecting mothers to surgery during pregnancy. Based on our experience, such a method overestimates true gestational age by up to 6 days.

Figure 26

Fig. 26. Summary of the maturation of retinal ganglion cells and their projection to the dorsal lateral geniculate nucleus from the 20th day of gestation to 2 months after birth. The horizontal bars mark the approximate duration of events: black portions of these bars represent periods of peak intensity. References: 1. Walsh et al.( 1983); 2. Hickey and Hitchcock (1984); 3. the present study; 4.Shatz (1983); 5. Shatz (1983), Chalupa and Williams (1985); 6. Cragg (1975), Winfield et al. (1980), Mason (1982), Shatz and Kirkwood (1984); 7. Cragg (1975), Greiner (1980), Morrison (1977, 1982); 8. Greiner (1980); 9. Moore et al. (1976) and this study; 10. Greiner (1980); 11. Sherman and Spear (1982); 12. Hubel and Wiesel (1970); 13. Mason (1982a,b), Sur et al. (1984).


 

Rise and fall of axon number in relation to the development of retinal projections

One axon per retinal ganglion cell. We have recently shown that axons do not branch in the nerve at early stages of development (Lia et al., 1985) and thus that the loss of axons in the fetal cat’s optic nerve is not due to the elimination of axon collaterals. Nor is there evidence that ganglion cell axons branch to any significant degree before they reach the optic nerve in several other species. In a study of 340 embryonic chicken retinal wholemounts stained by the pyridine-silver technique, Goldberg and Couloumbre (1972) saw very few branched axons, and in these few cases the branches extended only 5 to 10 µm away from the cell body. Similarly, Hinds and Hinds (1974) have shown that branching in the fiber layer of the embryonic mouse retina is extremely rare. Moreover, centrifugal and retino-retinal fibers (Bunt and Lund, 1981) probably do not contribute significantly to the elevated fiber population in the optic nerve of the cat. The presence of centrifugal fibers can be ruled out almost entirely since labeled cells have not been seen after large intravitreal injections of horseradish peroxidase in numerous fetal cats (eg., Williams and Chalupa, 1982; Shatz, 1983). While a retino-retinal projection appears to exist in the fetal cat, the number of cells involved is very small, well under 1% (B. Lia and L. M. Chalupa, unpublished; C. J. Shatz, personal communication). In sum, we feel justified in concluding that axons of ganglion cells do not branch, or branch only rarely, before the chiasm, and hence that there is close to a one-to-one correspondence between axons in the optic nerve and retinal ganglion cells in the cat at all stages of development. There is, of course, one caveat: that during the peak of neurogenesis the number of axons in the optic nerve will naturally lag slightly behind the number of young ganglion cells because the most newly generated axons will not yet have grown through the nerve (Lia et al., 1985). Our conclusion is that nearly 5 out of 6 ganglion cells in the cat’s retina are lost early in development.

Axon production and loss in relation to the genesis of retinal ganglion cells. Tritiated thymidine studies of ganglion cell neurogenesis in the cat have demonstrated that the first cells are generated in central retina before E-21 (Walsh et al., 1983; Walsh and Polley, 1985) and that the last cells are generated after E-35. An analysis of the time-course of axon ingrowth into the nerve provides an independent means to assess the period of ganglion cell production. Our results demonstrate that ganglion cells are generated as early as E19 and possibly as late as E39. Since the thymidine studies were based on an analysis of mature retinas, these results actually apply only to that small fraction of ganglion cells that survive to maturity. In contrast, our admittedly less direct method of assessing ganglion cell generation has the advantage of also taking into account those cells that ultimately die. The good correlation between these two very different methods implies that the population of cells that survive are generated within the same broad time period as those that are ultimately lost.

The central-to-peripheral distribution of growth cones we have described in the nerve could be a consequence of spatio-temporal gradients of ganglion cell generation (Walsh and Polley, 1985). If correct, it is possible that retinotopic order is retained in the nerve and that axons of neighboring ganglion cells grow along one another despite differences in time of generation. This seems unlikely, because growth cones in the optic nerve do not track along pre-existing fibers—consequently axons fail to retain neighbor relations (Williams and Rakic, 1985)—and because topographic order in the optic pathway is remarkably poor in adult cats (Horton et al., 1979). At present we simply lack a good explanation for the gradient in growth cone density in the optic nerve.

Axon loss and target innervation. Ganglion cell axons first reach the posterior thalamus by E28 (Shatz, 1983), at a stage when many neurons destined for the dorsal lateral geniculate nucleus are still migrating outward from the ventricular zone (Hickey and Hitchcock, 1984). The ingrowth of optic axons into their target nuclei is at least roughly concurrent with the earliest age at which we have demonstrated dying fibers in the optic nerve (Fig. 26). A similar correlation between the onset of fiber loss and the arrival of fibers among target cells has also been found in several motor systems (Hughes, 1965; Prestige, 1967; Hamburger, 1975; Landmesser and Morris, 1975; Landmesser and Pilar, 1976) and of the isthmo-optic projection of chick embryos (Clarke and Cowan, 1976). Collectively, these observations support a hypothesis, most forcefully advanced by Hamburger and Oppenheim (1982), that those fibers that die do so because they are unable to compete effectively for trophic molecules released in target tissues. Two observations, however, make us doubt whether the earliest fiber loss is actually related to axon-target interactions. First, we found necrotic axon terminal bulbs in the optic nerve. Clearly, this observation, if correct, is difficult to reconcile with the idea that axon loss is related to interactions with target cells. Second, even as late as E-38, the density of fibers in the dorsal lateral geniculate nucleus and superior colliculus is low (Williams and Chalupa, 1982, Shatz, 1983, Chalupa and Williams, 1985), extensive regions have only sparse input, and synaptic contacts of any sort are rare (Shatz and Kirkwood, 1984). These observations are also difficult to reconcile with the idea that fiber loss is due solely to competitive interactions between axons for trophic molecules.

At the time the peak population of axons is reached (E39), the retinal projection to the dorsal lateral geniculate nucleus, pretectum, and superior colliculus is still sparse. However, over the following week the density of the retinal influx becomes greater, and as early as E47 virtually all parts of every retino-recipient nucleus contain a heavy input of retinal axons (Williams and Chalupa, 1982, 1983a; Shatz, 1983; Chalupa and Williams, 1984). Paradoxically, during this same period when the number of axons in the nerve drops by approximately 300,000, the density of innervation, as assessed by anterograde tracing methods, is on the rise. Depending on one’s bias, these results can either be interpreted as showing that fiber loss is unrelated to the formation of retinal connections, or that the competition for trophic molecules during this period is so fierce that many ganglion cells are unable to survive.

Axon loss and segregation. Segregation between axons from right and left eyes begins in the dorsal lateral geniculate nucleus at E-46 (Shatz, 1983; Chalupa and Williams, 1984, 1985) and in the superior colliculus and pretectum about a week later (Williams and Chalupa, 1982, 1983a). Thus, the bulk of axons, some 400,000 fibers, are eliminated at least one week before the segregation of retinal projections is initiated. Indeed, the period when the most remarkable transformations in retinal projections are taking place corresponds to the slow phase of axon loss during which 50,000 to 150,000 fibers are lost. One of our principal reasons for examining fluctuations in the axon population was to determine to what degree axon loss could account for the formation of ocular dominance domains in the lateral geniculate nucleus and the superior colliculus. We conclude that while the loss of 50,000 to 150,000 axons could readily underlie the segregation of retinal projections, a large majority of fibers are eliminated for entirely different reasons (see the discussion of Chalupa and Williams, 1985). The relatively limited role of binocular competition in the loss of axons from the optic nerve of the cat is also demonstrated by the finding that the removal of one eye at a fetal age when the fiber population within the optic nerve is near its peak results in only a 20% increase in the number of fibers in the remaining optic nerve at maturity (Williams et al., 1983; Chalupa et al., 1984).

Axon loss and synpatogenesis. Besides segregation, two other major events occur during the last two weeks of gestation (Fig. 1B): First, the tempo of formation of retinal synapses increases greatly, and as early as eye opening the number of retinogeniculate synapses has been reported to be substantially greater than in the adult geniculate (Winfield et al.,1980). Second, ganglion cell dendrites form synapses with amacrine and bipolar cells (Cragg, 1975; Morrison, 1977, 1982; Greiner, 1980). Although there are important gaps in our knowledge of the kinetics of synapse formation in the cat’s visual system (and particularly the extent of turnover of early formed synapses), there is now enough evidence to conclude that approximately 100,000 retinal fibers are lost during the period when ganglion cells make the great majority of their synaptic contacts.

Axon loss and postnatal maturation of the visual system. The eyes of the kitten usually open during the early part of the second week, at about the same time that the first sluggish visual responses can be recorded from neurons in the superior colliculus (Stein et al., 1973), dorsal lateral geniculate nucleus (Adrien and Roffwarg, 1974; Daniels et al., 1978; Beckmann and Albus, 1982), and visual cortex (Hubel and Wiesel, 1963; Albus and Wolf, 1984). We have shown that even several days after eye opening (P12), about 100,000 axons still remain to be eliminated, and this raises the interesting possibility that the number of axons lost during this period may be affected by visual experience and ganglion cell activity. Our data and those of Z. Henderson (personal communication) demonstrate that the adult ganglion cell population is reached by the 6th week. Thus, the final population size is probably reached more than a month before the arbors of X- and Y-type retinal efferents have attained their characteristic dimensions (P56 to P90; see Mason, 1982a; and Sur et al., 1984), several weeks before the segregation of ocular dominance columns in striate cortex is complete (ca. P49; LeVay et al., 1978), and probably just before to the onset of the critical period (Hubel and Wiesel, 1970; Sherman and Spear, 1982).

A comparative and evolutionary perspective on ganglion cell death

The overproduction of retinal ganglion cells and axons appears to be a fundamental characteristic of the development of the mammalian visual system. Overproduction of ganglion cells has also been reported in the only avian species examined to date, the chicken (Rager and Rager, 1976; Rager, 1980). A common denominator in the development of mammals and birds is that the proliferation of ganglion cells ends either before or shortly after birth. In contrast, in those cold-blooded vertebrates in which ganglion cells are generated throughout life (Straznicky and Gaze, 1974) and in which the gradual increase in this population are accompanied by compensatory modifications of retinal connections (Gaze et al., 1974; Scott and Lazar, 1976; Easter and Stuermer, 1984; Reh and Constantine-Paton, 1984), there is no overproduction of optic fibers whatsoever (Gaze and Peters, 1961; Wilson, 1971; Easter et al., 1981; Dunlop and Beazley, 1984). Evidently, there are two strategies to arrive at suitable matches between the population of ganglion cells and the population of target cells. In mammals and birds a certain degree of flexibility during the formation of visual connections is achieved by producing more neurons than are ultimately needed (cf. Cowan, 1973), whereas in other classes—e.g., fish and amphibia—the system is malleable throughout life, thereby making the production of excess neurons or axons unnecessary.

However, it remains a puzzle why there should be such sizable differences in the degree of overproduction of ganglion cell axons in warm-blooded vertebrates. Why should there be a 5- or 6-fold overproduction in the domestic cat, a 2- or 3-fold overproduction in primates (Rakic and Riley, 1983a; van Driel and Provis, 1983), and substantially less than a 2-fold overproduction in the chicken (Rager, 1980)? Rakic (1985) has pointed out that those species with extensive fields of binocular vision and a greater uncrossed retinal projection—in particular, humans, rhesus monkeys, and cats—appear to loose a larger proportion of fibers, and that the severity of axon loss may depend on the degree of intermingling and competition between axons from right and left eyes at early stages of development. In support of this idea, removal of one eye at early stages in different species (Rakic and Riley, 1983b; Williams et al., 1983; Chalupa et al., 1984; Sefton and Lam, 1984) reduces axon loss in the remaining optic nerve in proportion to the extent to which axons from right and left eye normally intermingle during development (Rakic, 1985). However, it is clear that binocular competition does not explain the bulk of axon overproduction in most species, and for instance in the cat, the termination of binocular competition spares considerably less than one-tenth of those axons normally lost.

In a similar vein, it has been suggested that the loss of neurons and axons is a consequence of the elimination of topographically or functionally inappropriate connections (McLoon, 1982; Jeffery and Perry, 1982; Williams et al., 1983; Insausti et al., 1984; Jeffery, 1984; Chalupa and Williams, 1984, Jacobs et al., 1984). Although errors certainly do occur in small numbers during development, it seems unlikely that the degree of ganglion cell overproduction in different species is proportional to the imprecision of retinal connections. To be specific, it is unlikely that initial retinal connections in cat are so imprecise in comparison to other species that the most effective solution is to build in a 5– or 6–fold safety factor. In fact, recent results suggest that pattern of retinal projections, particularly the distribution of crossed and uncrossed fibers at the chiasm, are actually remarkably precise in fetal cats (Shatz and Kliot, 1982; Lia et al., 1983).

Wildcat Illustration

It is possible that differences in the magnitude of overproduction of ganglion cells and their axons have to do with the evolutionary trends of different species (cf. Katz and Lasek, 1978; Albrech et. al., 1979). During the late Pleistocene and particularly during the last 10 millennia, the body size of the species from which the domestic cat is derived—Felis silvestris—has become radically smaller (Kurtén, 1965; Kurtén, 1968; Hemmer, 1974). As little as 15,000 years ago Felis silvestris was more than twice as massive as either the domestic cat or most extant European and North African wildcats. Because brain size is proportional to body size—in non-primate mammals the correlation (or allometric coefficient) between these variables is about 0.74 (Jerison, 1973; Martin, 1981; Eisenberg and Wilson, 1982)—the recent reduction in the body size of the cat has probably been associated with a corresponding reduction in the size of the brain, eye, and retina and in total neuron number. Thus, some fraction of the ganglion cell excess in the fetal cat may be a phyletic holdover, and the elimination of this fraction may correspond to what Glücksmann (1951) refers to as phylogenetic cell death. In essence we are arguing that the rapid reduction in the size of the cat has been achieved by changes in the developmental program that only take effect at comparatively late stages, well after ganglion cells have been generated. Although admittedly speculative, this idea can be tested: If correct, it follows that closely related, and much larger feline subspecies, particularly the Spanish wildcat, Felis silvestris tartessia, should produce approximately the same number of retinal ganglion cells but fewer of these should be lost at later stages of development, and as a consequence the ganglion cell population at maturity should be greater than in the domestic cat.

[This hypothesis was subsequently confirmed by Williams, Cavada, and Reinoso-Suárez, in the paper Rapid Evolution of the Visual System published in The Journal of Neuroscience in 1993.]

Converging lines of evidence now indicate that multiple factors—among them axon-target interactions, competition for limited resources by either axons or dendrites, and autonomous programs of ganglion cell development—regulate the severity of cell and fiber loss in the mammalian and avian visual system. The substantial species differences provide clues to the relative importance of this multitude of factors and may ultimately give us insight into the purpose of cell and fiber overproduction in brain development and evolution.

 

ACKNOWLEDGMENTS

This study was supported by NIH grant NEI RO1 EY-3391 to LMC. We thank Deborah van der List for expert technical assistance.


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