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Development of the optic nerve of the opossum (Didelphis virginiana)
Developmental Brain Research, 44 (1988) 37-48 Elsevier
37
BRD 50825
Development of the optic nerve of the opossum
( Didelphis virginiana) Michael A. Kirby 1, Paul D. Wilson 2 and Thomas M. Fischer 2 IDepartments of Pediatrics and Anatomy, School of Medicine, Loma Linda University, Loma Linda, CA 92350 (U.S.A.) and 2Department of Psychology, University of California, Riverside, Riverside, CA 92521 (U.S.A.) (Accepted 5 July 1988) Key words: Development; Optic nerve; Enucleation; Re-organization
The development of the optic nerve of a marsupial, the North American opossum, was examined in 24 animals from postnatal days 5 to 78 (P5-P78); gestation is 13 days. The estimated number of axons increased from 24,000 at P5, to 267,000 at P27, approximately 2.7 times the mean number in the adult. Following P27, axon numbers decreased rapidly to 140,000 at P40, then decreased more slowly, attaining adult values between P50 and P59. Thus, the opossum is similar to placental mammals examined in evidencing an overproduction and later attenuation to adult values in the number of axons in the optic nerve during development. Monocular enucleation of 3 animals at P17, 10 days before peak axon counts, resulted in a mean population increase of 24,000 (range 19,000-30,000) above the normal adult mean. Additionally, a 4th animal monocularly enucleated on P7, 3 days prior to the arrival of migrating fibers to central target sites, had a similar value of 26,500 supernumerary axons. Our findings in the opposum, when coupled with previous reports in other mammals, suggest that binocular interactions during development account only for optic nerve axon loss approximately equal in magnitude to the ipsilateral projection from one eye.
INTRODUCTION Initial o v e r p r o d u c t i o n and subsequent reduction of axon n u m b e r s in the optic nerve during development have been o b s e r v e d in the rat t°'14As'25,32, hamster 48, rabbit 42, marsupial native cat 11 and q u o k k a 3 (although see ref. 1), m o n k e y 38, human 37 and cat 30'49, and have been c o r r e l a t e d with the overproduction and elimination of retinal ganglion cells in the rat 25'32,36, rabbit 42, h a m s t e r 44, and cat 7. While all the contributing mechanisms are not fully understood, it has been suggested that this p h e n o m e n o n , the substantial reduction in axon numbers, reflects competition for terminal synaptic space 7'12'31'49. The evidence that binocular competition is an i m p o r t a n t mechanism contributing to the reduction of axon numbers in the developing optic nerve derives principally from studies d e m o n s t r a t i n g that m o n o c u l a r enucleation
near the time when axon counts p e a k reduces the severity of axon loss in the remaining optic nerve. Counts of axons in the remaining nerve of mature animals monocularly enucleated during early develo p m e n t evidence little or no increase above normal in the rat 43, 25% increase in the cat 49, and 34.7% increase in the m o n k e y 39. A t present, the reasons for this species difference is not understood. Previous investigators 39'43 have n o t e d that the magnitude of the increase in axon n u m b e r s is related to the size of the ipsilateral population in each of these species. The rat has a small ipsilateral projection that averages less than 4 % of the total ganglion cell population in p i g m e n t e d animals 15,2° and less than 2% in albinos 15'43, c o m p a r e d to a p p r o x i m a t e l y 28% in the cat (ref. 47, p. 283) and 40% in the m o n k e y 34. The opossum has a substantial ipsilateral projection (approximately 20% of the ganglion cell population, see ref.
Correspondence: M.A. Kirby, Department of Pediatrics, School of Medicine, Loma Linda University, Loma Linda, CA 92350, U.S.A. 0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
38 23, Fig. 1; personal observations) and offers the opportunity to examine the magnitude of increase in the number of optic nerve axons following early monocular enucleation in an animal that has an ipsilateral projection intermediate in size between the rat and cat. Most studies which have examined the effects of disruption of binocular competition during development have removed one eye near the time when the number of axons has peaked. This opens the possibility of confounding the consequences of disruption of binocular competition with the effects of axon degeneration or denervation of central target sites 45. Monocular enucleation performed prior to the arrival of retinal afferents at central target sites provides an opportunity to distinguish the effects of binocular competition from the effects of denervation or degeneration at central targets. In the present study we have examined the normal development of the optic nerve and the effects of early monocular enucleation on axon counts in the remaining optic nerve of the mature animal enucleated either 10 or 20 days before optic nerve counts normally peak. As a marsupial, the opossum is an ideal species for the study of early neural development. Opossum pups are accessible in the pouch at a relatively young age (born following 13 days gestation, ref. 41), and remain in the pouch until their eyes open at about 60 days after birth. A partial report of these findings has been given 24. MATERIALS AND METHODS
Experimental animals Twenty-four pouch-young animals, obtained from 7 litters, were used in the present study. Postnatal ages were established from known birthdates (2 litters), or estimated (5 litters) by body weight and snout-rump lengths using the method of Cutts et al.~3. Known birthdates were established by checking daily the maternal marsupium for the presence of young, the appearance of which was counted as postnatal day I (P1). The ages obtained from these observations agreed closely with the calculated age using body measurements ~3.
Histologicalprocedures Following either i.p. injections of sodium pento-
barbital or hypothermia-induced anesthesia, animals were perfused through the heart with buffered phosphate (pH 7.6), followed by a mixture of 4% paraformaldehyde 2% glutaraldehyde in the same buffer. The optic nerves with the globe attached for orientation were then removed and stored at room temperature in the fixative for 12-24 h, a step found to be critical for the preservation of the uitrastructure of the individual axons. Care was taken during the removal of the tissue to minimize physical distortion and to prevent desiccation by bathing the tissue with buffered fixative during dissection. The tissue was then postfixed in 1-2% osmium, washed in several' changes of buffered phosphate, dehydrated through an ascending series of acetone, and embedded in Spurr's plastic. Within each block, the optic nerve was carefully oriented to insure that all sections saved for analysis were cut directly transverse to the long axis of the nerve. Since axons may extend through the nerve at different times and with varied rates of growth, the numerical data obtained at a particular age and the measures of changes across ages could be substantially affected if samples were taken at variable distances along the nerve. To minimize such problems, the ultrathin (grey to grey-silver) and semithin (gold to purple) sections were obtained from each nerve at 1.2-2.5 mm from the globe. Compression in the ultrathin sections was relieved by xylene vapor and in the semithin sections by mild heat. Ultrathin sections were mounted on copper mesh grids (#300), stained with uranyl acetate and lead citrate, and photographed on either a Hitachi H-500 or H-600 electron microscope. Contrast for the youngest tissue was improved by prolonged staining times (2 h) in the uranyl acetate.
Sampling procedure Critical to the study was the use of a sampling procedure that would uniformly cover each nerve crosssection. For each nerve, electron micrographs were taken within each grid opening containing neural tissue, and for most nerves, 4 adjacent but non-overlapping micrographs were taken within each grid opening. For the late postnatal nerves (>P50) the number of grid squares available was greater and only two micrographs in diagonal corners of each grid square were obtained. The areas photographed were chosen
39 without regard for the relative amounts of the region occupied by axons or glia, but with consistent reference to the grid bars. Large blood vessels were not photographed. This type of sampling procedure was shown earlier 22 to sample all regions of the nerve uniformly and is similar to the method of Williams et al. 49'5° (see ref. 50 for a discussion of the advantages of this sampling technique). Electron micrographs were taken at a magnification of approximately 5000x, and enlarged in printing to a final magnification of 15,000-16,000x. Care was taken to ensure consistent magnification through the use of a calibration grid (Pelco #606). For the first sets of nerves the calibration grid was photographed at the beginning and end of each session. Since in each case the two measurements agreed within 0.1%, the calibration grid was photographed only once for each filming session for the remaining nerves. The total number of neural processes (axons and growth cones) in each nerve was estimated by counting the number of processes in the areas sampled in the ultrathin section and extrapolating to the entire cross-sectional area of the nerve (minus the large vascular elements) measured from the adjacent semithin section. No correction for differential compression was used, given evidence in a previous study 22 and in the present material that area differences between adjacent ultrathin and semithin sections were consistently less than 4%, and typically 1-2%. A random sample of neural processes for area measurement was obtained by drawing a diagonal line across each micrograph and measuring all processes which came in contact with the line. Myelin sheath when present was excluded in the measurement. Measurements of the processes were made on a computer-linked digitizer (Houston Instruments Hipad), and the area of each process was computed using a morphometry program (Bioquant II, R & M Biometrics). Distributions and descriptive statistics were generated using an SAS statistical package. Monocular enucleation
Optic nerve counts were obtained from 4 animals monocularly enucleated at P17 or P7. The female (containing the pouch young) was anesthetized (40 mg/kg Nembutal, i.p.) and the marsupium gently
opened to permit access to the young. Following administration of topical anesthetic (benzocaine), theeyelids of each pup were gently parted and the globe rotated free of the socket and removed. The socket was then repeatedly flushed with antiseptic and anesthetic (iodine and benzyl chloride), and the marsupium closed. For each animal the anesthetic level appropriate for enucleation was sufficient to eliminate all reflexive movement in response to a forceps pinch to the eyelids and surrounding tissue, and was attained 2-3 min after anesthetic application. RESULTS Optic nerve processes
Figs. 1 and 2 illustrate examples of the micrographs taken from the optic nerve of animals at different ages. The axons of young opossums, like those of adult opossums z2, could be distinguished from surrounding glial elements by the presence of microtubules and neurofilaments, as well as by the relative translucence of the axonal cytoplasm. Even in the youngest animals examined (P5, P9 in Fig. 1) the translucent quality of the axoplasm contrasted sharply with the darker glial processes which contained numerous fine fibrils and ribosomes as described previously for the opossum z2 and other mammals 35,5°. Also present in the youngest nerves were relatively large, irregular processes that resembled the axons in their subcellular components, but were presumed to be growth cones on the basis of their similarity to processes identified in the cat and monkey5°,51. These processes occasionally contained numerous organelles (i.e. clear vesicles, microfilaments, endoplasmic reticulum) characteristic of the bulbous central core of neural growth cones 5°. The majority of these processes had few of these complex subcellular components and were composed principally of a mesh of fine microfilaments in a translucent cytoplasm, similar in appearance to the most distal portions of neural growth cones in the cat and monkey5°'51. In the present study, the profiles presumed to be growth cones clearly differed from glial processes by characteristics which distinguished axons from glia, and were identified in the youngest nerves examined (e.g. P9, P10 in Figs. 1 and 2). Occasionally axon profiles were encountered that contained either darkly mottled cytoplasm or an ir-
m (gestation in this species is 12.5 days). Pres,inr ...nrn t.m;nn,,.. I,“,., ;r
41 regular membrane, but were otherwise surrounded by axons that were normal in appearance. Such profiles are morphologically similar to necrotic axon profiles described in the developing optic nerve of the c a t 49 (see also ref. 9), rat 43, and monkey 38. These processes were observed in the youngest ages examined, suggesting that some axon loss from the putative nerve occurs very early, even before the majority of migrating neurons reach central target sites. Although these observations are consistent with findings in the fetal cat 5°, our inability to identify these processes conclusively as necrotic (e.g. with enzyme markers, pyknotic nuclei) prevented a quantitative analysis of their numbers. What is clear is that very few of these profiles could be identified at any stage of development, suggesting that axon loss (through cell death or retraction) is a rapid process not readily identified with routine electron microscope techniques.
Axon numbers Fig. 3 illustrates the calculated axon population in each optic nerve plotted as a function of postnatal age. At P5, the axon population was estimated at 24,000, approximately 25% the mean adult value of 100,000. Over the next 3 weeks the number of optic nerve fibers increased rapidly, reaching 250,000 by the end of the third postnatal week. Maximum counts were obtained in our series at P27, with an estimated optic nerve fiber population of 267,000, approximately 2.7 times the mean adult value. Over the next few days the number of axons decreased to approximately 240,000 by P31 and 140,000 by P40. Following P40, axon counts gradually decreased and reached adult values between P50 and P59. To investigate the reliability of our sampling procedure, both optic nerves were counted in 5 animals. At P30 this variability was 3.0% (252,875 vs 245,188) and 3.4% (239,124 vs 230,975). In the remaining ani~ mais, the oldest examined (Fig. 2), the variability between the two nerves ranged from 4.2% (106,993 vs 102,498) to only 0.4% (110,679 vs 110,273) and 0.3% (107,843 vs 107,482). Since variance between the two nerves is equal or less than that observed when the same adult nerve is sectioned in two different locations e2, we feel fairly confident that our sampling procedure provided a reasonable degree of reliability.
Disruption of binocular competition To examine the effects of binocular competition on the remaining optic nerve, 3 opossum pups were monocularly enucleated on P17, 10 days prior to peak axon counts in normal animals. Retinal fibers first invade the thalamus on P10 (refs. 5, 6, 29) in D. marsupialis aurita, a species closely related to and having the same gestation period (12.5 days) as D. virginiana. The possibility exists that enucleation at P17 may cause axonal degeneration and denervation within central target sites, which might affect the number of axons in the remaining nerve, thereby potentially confusing secondary effects of denervation and degeneration with binocular competition 45. To minimize this possibility, one additional animal was enucleated on P7, which is approximately 3 days prior to arrival of fibers at their central target sites in D. marsupialis aurita and 20 days prior to peak axon counts found in this study. Counts were obtained from the remaining optic nerve of these 4 animals between P75 and P78, at which time the fiber population in normal animals is within the adult range. The mean fiber population for the P7 enucleate (126,508) was not substantially different from the P17 enucleates (119,231; 122,000; 129,790). The mean population for the 4 enucleates was 124,382 compared to 103,854 in 5 age-matched controls (range 94,271-110,679; Fig. 2).
Axon size Although the presumed growth cones were often markedly larger in area than axons (Fig. 1), the distribution of the cross-sectional areas of the two were continuous, as has been observed in other species (e.g. refs. 43, 50). Rather than attempting to distinguish between growth cones and axons of similar cross-sectional area on the basis of subcellular components and morphology, we included growth cones in the measurement of axons. Fig. 2 illustrates the axons (and growth cones when present) from the optic nerve of opossums at various ages. Cross-sectional areas ranged 0.001-1.21 /~m2 from P5 to P18. At later ages cross-sectional areas diminished and were 0.002-0.53/~m 2 at P24 and P27. The loss of profiles greater than 0.6/~m 2 in area paralleled an apparent decrease in growth cones which were frequently observed from P5 to P10, but rarely encountered in micrographs at P24 and P27. Following P27, the range
42
43
P75
Adult Fig. 2. Electron micrographs from the optic nerve of opossum pups at postnatal ages 10-75 (P10-P75) and the adult. At early ages numerous profiles were present, presumed to be growth cones, suggesting the continuing addition of axons to the developing optic nerve. At P27, the age having peak counts in our material, few growth cone processes were encountered. By P59, individual axons were fewer in number overall, and had larger cross-sectional areas (large arrow denotes early myelinated process). At P75 the majority of profiles were myelinated (large arrow), although several unmyelinated processes were still present (small arrows). These were smaller in cross-sectional area and had relatively less myelination than encountered in the adult. Bar = 1/~m.
of axon sizes increased to 0.003-1.69 pm 2 in area at. P50 and 0.038-2.94pm 2 at P78. Axons in the remaining nerve of 4 enucleated animals at P75-P78 were 0.018-3.55 pm 2 in area, similar to those in normal animals. Although the total number of axons in the nerve at P59-P78 is within the range found in the adult, the largest axons found at P78 were still considerably smaller in diameter than those in the adult. In adult optic nerve 22, axons excluding myelin are 0.12-6.06 pm 2 in diameter (equivalent to 0.01-28.8 /~m2 in area, if profiles are round). Apparently the
major increase in axon caliber occurs after the period of axon loss and the attainment of adult numbers. Correlated with developmental increase in the diameter of axons was the onset of myelination. The first myelinated fibers were present in our material at P50 when axon numbers were near normal for the adult; less than 2.0% were myelinated at this age. Thereafter, the number of myelinated fibers began to increase rapidly; 11% of the fibers were myelinated by P59 and 34% by P78. This is consistent with the finding in other mammals that myelination of optic fi-
44 bers begins lized30,38.43
when
axon
numbers
have
stabi-
Growth of the optic nerve The size of the optic nerve increased progressively with postnatal age. At P9 the cross-sectional area was only 0.009 mm 2, less than 2.4% of the mean adult area of 0.38 mm 2 reported previously 22. Present in the nerve at early postnatal ages but absent in the adult were numerous septa that divided the optic fibers into discrete fascicles. The size of each fascicle was not correlated with its position within the nerve; fascicles containing large numbers of axons were just as numerous near the center of the nerve as towards the periphery. The number of fibers within each fascicle varied considerably; however, in general the number of processes within each fascicle initially increased at early ages until P27. At P5, individual fascicles contained only 20-40 fibers whereas by the end of the third postnatal week individual fascicles contained 100-200 fibers (Fig. 2). From the end of the third postnatal week on, the number of processes within each fascicle decreased until by P50 only 10-20 axons were present within each fascicle. By P59 no fasciculation was apparent, which is characteristic of the adult opossum. The total nerve crosssectional area at P50 was 0.071 m m 2 (19% of the adult size 22) and 0.11 mm 2 (30% of adult) at P78.
,300,000 t 250,000J m
0(D
O
200,000
z0 150,000 X <:(
1oo,00o
! 8
50,000 0 ' 1'0' 2'0' 3'0" 4'0" 5'0' 6'0" 7'0 8'0 POSTNATAL ACE (DAYS) Fig. 3. Plot of the estimated population of axons in the developing optic nerve of the opossum at different postnatal ages.
Filled circles denote 3 animals monocuIady enucleated during early postnatal development (see text).
DISCUSSION
Changes in axon number with development This study has described an initial increment and later attrition in the number of axons within the developing optic nerve of the North American opossum. Similar to the growing list of placental mammals, e.g. monkey 38. cat 3°'49. rabbit 42. hamster 48. rat 1°'25'32. human 37, the development of the opossum optic nerve exhibits successive phases of development. The first phase, probably starting near birth (at E12.5) and continuing to the end of the 4th postnatal week, is characterized by a rapid increase in the number of axons within the presumptive nerve. A peak value of approximately 270,000 axons. 2.7 times normal adult values, is attained by P27. This is followed by a second phase of relatively rapid axon loss. Bv P40 the number of axons has decreased to approximately 127,000, roughly 30,000 axons more than the average number in the adult. In the third phase, axon numbers decrease more slowly to reach normal adult values by about the 8th postnatal week. Thus axon numbers of this marsupial exhibit a development pattern quite similar to that seen in placental mammals. Technical considerations Consideration must be given to several technical problems that could potentially affect our observations. The first concerns the possible inclusion in our counts of multiple growth cone processes that originate from a single migrating fiber. In culture, the distal portions of axons divide into multiple filopodial extensions 4'8, and the use of a single cross-section through the developing nerve may result in an overestimation of the actual fiber population during periods where numerous growth cone processes are present. Recent observations by Mason (refs. 2, 27, and personal communication) indicate that the terminal morphology of growth cones is less complex within fiber pathways in vivo than within either target nuclei in vivo or preparations in vitro, suggesting that multiple growth cone processes may be less of a problem than would be predicted on the basis of the structural complexity observed in vitro. Furthermore, since the total number of axons increased almost linearly between P5 and Pt8 when apparent growth cone profiles were decreasing in number, the possible inclusion of multiple processes from the same cell proba-
45 bly contributed little to the estimate of the total axon population in this period. The developing optic nerve of the quokka has been reported to have axon numbers which greatly exceed the total estimated number of retinal ganglion cells and it has been suggested that optic axons may branch extensively in the quokka optic nerve during early development, with subsequent loss of collaterals later in development 3'16. However, the presence of branching profiles within the nerve may reflect fixation artifact, and the discrepancy between axon and ganglion cell counts may have resulted from the inability to label the entire ganglion cell population through retrograde transport techniques. We have succeeded in labeling 75% of the retinal ganglion cell population predicted from nerve axon counts in a P30 opossum (unpublished observations). Equivalent or higher correlation has been obtained between counts of nerve axons and cells in the ganglion cell layer during development in rat 32, rabbit 42, and cat (Lia and Chalupa, personal communication). Recently, Lia et al. 26, counting axons at the proximal and distal ends of the fetal optic nerve of the cat, found more axons near the eye than near the chiasm, arguing against significant bifurcation of axons within the nerve during development. A second potential problem concerns the method by which the total population of each nerve was estimated. We have shown previously in the adult 22, that sampling all grid squares containing neural tissue in a single cross-section reduces the chance for error arising from changing axon densities associated with axons of different diameters and glial processes. However, this method when employed in the relatively small nerves of the pouch-young opossum may introduce other problems (e.g. adequate sample area, coverage of neural processes by grid bars). To evaluate this possibility we analyzed both nerves in each of 5 normal animals. The difference in counts between the two nerves in these animals varied from 0.3 to 4.2% (Fig. 3). Thus we feel confident that this technique permits a relatively accurate assessment of the total population within the nerve of individual animals for both normal an enucleated animals. It is important to point out that the addition of axons to the developing nerve may be accompanied by simultaneous axon loss, although we could not identify with certainty necrotic axon profiles. The exis-
tence of numerous macrophages in the optic fiber layer of the retina as early as P22 (Kirby and Wilson, in preparation), several days before the sharp decline in axon numbers, supports the possibility that cell death begins well before the peak in retinal ganglion cell numbers has been obtained. Therefore, the actual number of fibers added to the developing nerve most likely exceeds the number reported here. Recently, Williams et al. 5° have calculated, from estimates of the number and time course of axon loss in the developing optic nerve of the cat, that approximately 20% of all axons are lostprior to the period of peak count. Taking into account the same ratio and time course for axon removal (1 h) used by Williams et al. 5° for the cat, the opossum optic nerve may have an actual peak fiber population as large as 324,000.
Development of retinal projections In close agreement with our findings on the development of the opossum optic nerve are the recent observations of the development of retinal projections to the superior colliculus of Didelphis marsupialis, a South American opossum 29. At P7 in this species, retinal fibers have reached only the rostral tip of the optic tract and the first retinal fibers arrive at the superior colliculus by P10. Retinal ingrowth to the superior colliculus continues to increase over the next 12 days, with maximum overlap between projections of the two eyes occurring on P22, a few days before the time of peak axon counts in the present study. Retraction of the exuberant ipsilateral projection to the adult pattern does not start until P26, which is approximately the time of the onset of greatest axon loss in the optic nerve in our material. The first laminar segregation of retinal terminals in the superior colliculus of D. marsupialis does not occur until P47, which is near the time period when axon counts in the optic nerve of D. virginiana attain adult values. Thus the time of ingrowth, expansion, and later retraction to the adult pattern of retinal afferents to the superior colliculus corresponds closely with our observed time-course of axon addition and later attrition in the optic nerve.
Effects of early monocular enucleation We have shown that early monocular enucleation in the opossum results in an increase above normal of approximately 24,000 axons in the remaining optic
46 nerve. This value corresponds to the number of ganglion cells estimated to project ipsilaterally in this species (unpublished observations) and suggests that elimination of binocular competition reduces cell death proportionate to the size of the ipsilateral projection. In the cat, which has an ipsilateral projection involving approximately 28% of the adult ganglion cell population 47, prenatal monocular enucleation increases the number of axons in the remaining optic nerve by 25% (ref. 49) and the number of ganglion cells in the remaining retina 7 of the adult. In the primate 39, which has 40% of the ganglion cell population projecting ipsilaterally34, monocular enucleation in the fetus increases the number of axons in the remaining nerve of the adult by 32-38%. In contrast to these two species, the size of the ipsilateral projection in the albino rat 15'43 is only 1% (approximately 1200 axons). Monocular enucleation during early development in this species does not result in an apparent increase in the number of axons in the remaining nerve of the adult25; however, normal interanimal variation in optic nerve numbers could presumably obscure any supernumerary axons that might result. The possibility that supernumerary axons may be present is suggested by observations that early enucleation results in greater than normal numbers of ganglion cells that project ipsilaterally in the remaining retina (e.g. refs. 17, 19, 20, 40). In each of these species, however, enucleation was performed at a time when ocular inputs from the two eyes had already reached central sites of termination, opening the possibility that factors other than binocular competition, e.g. re-routing at the chiasm, axonal degeneration, or relay cell denervation 19'45 might alter the numbers of fibers in the remaining nerve. To explore this possibility, we monocularly enucleated one animal at P7, prior to the ingrowth of retinal projections to central sites of termination. The number of axons in the remaining nerve of this animal did not differ significantly from the axon numbers in animals enucleated at P17. This is consistent with reports on
REFERENCES 1 Beazley, L.D. and Dunlap, S.A., The evolution of an area centralis and visual streak in the marsupial Setonix brachyurus, J. Comp. Neurol., 216 (1983) 211-231. 2 Bovolenta, P. and Mason, C., Growth cone morphology
the fetal cat, in which monocular enucteation during either early fetal periods (E23, refs. 45, 46) or relatively late 49 produces equivalent axon populations in the remaining nerve. Interestingly, in the cat 4s and rat ~9monocular enucleation at relatively early stages of retinal development (i.e. prior to the arrival of axons at the optic chiasm) results in a smaller than normal ipsilateral projection, suggesting that in addition to eliminating competition at central target sites, monocular enucleation may affect guidance of afferent fibers within the optic pathway, However, most findings to date imply that factors intrinsic to binocular competition at central target sites determine the magnitude of the supernumerary population, although other factors may also be important 21'33'45. Although speculative, this suggests that one effect of monocular enucleation is to increase the availability of terminal synaptic sites (or trophic factors) within the portions of retinorecipient structures having binocular representations, thereby maintaining cells that are otherwise lost through binocular competition. Some support for this is found in a recent observation that the majority (in absolute numbers) of supernumerary ganglion cells resulting from early monocular enucleation in the cat are located in the area centralis 7'21, where interactions during development between binocular competition and cell density would be maximum. The substantial body of evidence in the rat, mouse, hamster, and cat demonstrating the maintenance of an enlarged ipsilateral pathway in the mature animal following monocular enucleation during development would indicate that the principal effect of binocular competition is the retraction of retinal afferents coming from the inappropriate eye. It would be of interest to analyze ganglion cell receptive field properties and topographic organization (retinal location and number of ganglion cells) of central projection patterns for the remaining eye of adult animals monocularly enucleated during development.
varies with position in the developingmouse visual pathway from retina to first targets, J. Neurosci., 7 (I987) 1447- 1460. 3 Braekevelt, C.R., Beazley, L.D., Dunlap, S.A. and Darby, J.D., Number of axons in the optic nerve and retinal ganglion cells during development in the marsupial Setonix
47 brachyurus, Dev. Brain Res., 25 (1986) 117-125. 4 Bray, D. and Chapman, K., Analysis of microspike movements on the neuronal growth cone, J. Neurosci., 5 (1985) 3204-3213. 5 Cavalcante, L.A. and Rocha-Miranda, C.E., Postnatal development of retinogeniculate, retinopretectal, and retinotectal projections in the opossum, Brain Res., 146 (1978) 231-348. 6 Cavalcante, L.A., Rocha-Miranda, C.E. and Linden, R., Observations on postnatal neurogenesis in the superior colliculus and the pretectum in the opossum, Dev. Brain Res., 13 (1984) 241-249. 7 Chalupa, L.M., Williams, R.W. and Henderson, Z., Binocular interaction in the fetal cat regulates the size of the ganglion cell population, Neuroscience, 12 (1984) 1139-1146. 8 Connolly, J.L., Seeley, P.J. and Greene, L.A., Regulation of growth cone morphology by nerve growth factor: a comparative study by scanning electron microscopy, J. Neurosci., 13 (1985) 183-198. 9 Cooke, R.D., Ghetti, B. and Wisniewski, H.M., The pattern of Wallerian degeneration in the optic nerve of newborn kittens: an ultrastructural study, Brain Res., 75 (1974) 261-275. 10 Crespo, D.D., O'Leary, D.M. and Cowan, W.M., Changes in the numbers of optic nerve fibers during late prenatal and postnatal development in the albino rat, Dev. Brain Res., 19 (1985) 129-134. 11 Crewther, D.P., Nelson, J.E. and Crewther, S.G., Afferent input for target survival in marsupial visual development, Neurosci. Lett., 86 (1988) 147-154. 12 Cunningham, T.J., Mohler, I.M. and Giordana, D.L., Naturally occurring neuron death in ganglion cell layer of the neonatal rat: morphology and evidence for regional correspondence with neuron death in superior colliculus, Dev. Brain Res., 2 (1982) 203-215. 13 Cutts, J.H., Kruse, W.J. and Leeson, C.R., General observations on the growth and development of the young pouch opossum, Didelphis virginiana, Biol. Neonate, 33 (1978) 264-272. 14 Dreher, B., Potts, R.A. and Bennett, M.R., Evidence that the early postnatal reduction in the number of rat retinal ganglion cells is due to a wave of ganglion cell death, Neurosci. Left., 36 (1983) 255-260. 15 Dreher, B., Potts, R.A., Ni, S.Y.K. and Bennett, M.R., The development of heterogeneities in distribution and soma sizes of rat retinal ganglion cells is due to a wave of ganglion cell death. In J. Stone, B. Dreher and D. Rapaport (Eds.), Development of the Visual Pathways in Mammals, Liss, New York, 1984, pp. 39-57. 16 Dunlop, S.A. and Beazley, L.D., A retino-retinal projection contributes minimally towards high axon numbers in developing optic nerve of the marsupial Setonix brachyurus, Soc. Neurosci. Abstr., 12 (1986) 121. 17 Fawcett, J.W., O'Leary, D.D.M. and Cowan, W.M., Activity and control of ganglion cell death in the rat retina, Proc. Natl. Acad. Sci. U.S.A., 81 (1984)5589-5593. 18 Foster, R.E., Conners, B.W. and Waxman, S.G., Rat optic nerve: electrophysiological, pharmacological, and anatomical studies during development, Dev. Brain Res., 3 (1982) 371-386. 19 Godemont, P., SalaiJn, J. and M6tin, C., Fate of uncrossed retinal projections following early or late prenatal monocular enucleation in the mouse, J. Comp. Neurol., 255 (1987)
97-109. 20 Jeffery, G., Retinal ganglion cell death and terminal field retraction in the developing rodent visual system, Dev. Brain Res., 13 (1984) 81-96. 21 Kirby, M.A. and Chalupa, L.M., Retinal crowding alters the morphology of alpha ganglion cells, J. Comp. Neurol., 251 (1986) 532-541. 22 Kirby, M.A., Clift-Forsberg, L., Wilson, P.D. and Rapisardi, S.C., Quantitative analysis of the optic nerve of the North American opossum (Didelphis virginiana): an electron microscopic study, J. Comp. Neurol., 211 (1982) 318-327. 23 Kirby, M.A. and Wilson, P.D., Receptive field properties and latency of cells in the lateral geniculate nucleus of the North American opossum (Did@his virginiana), J. Neurophysiol., 56 (1986) 907-933. 24 Kirby, M.A. and Wilson, P.D., Axon count in the developing optic nerve of the North American opossum: overproduction and elimination, Soc. Neurosci. Abstr., 10 (1984) 467. 25 Lam, K., Sefton, J. and Bennet, M.R., Loss of axons from the optic nerve of the rat during early postnatal development, Dev. Brain Res., 3 (1982) 487-491. 26 Lia, B., Williams, R.W. and Chalupa, L.M., Overproduction of optic nerve fibers in the fetal cat does not reflect axon branching within the nerve, Suppl. Invest. Ophthaltool. Vis. Sci., 26 (1985) 286. 27 Mason, C.A., Growing tips of embryonic cerebeUar axons in vivo, J. Neurosci. Meth., 13 (1985) 55-73. 28 Mason, C.A., How do growth cones grow?, Trends Neurosci., 8 (1985) 304-306. 29 M6ndez-Otero, R., Cavalcante, L.A., Rocha-Miranda, C.E., Bernardes, R.F. and Barrados, P.C,R., Growth and restriction of the ipsilateral retinocular projection in the opossum, Dev. Brain Res., 18 (1985) 199-210. 30 Ng, A.Y.K. and Stone, J., The optic nerve of the cat: appearance and loss of axons during normal development, Dev. Brain Res., 5 (1982) 263-271. 31 Oppenheim, R.W., Neuronal cell death and some related regressive phenomena during neurogenesis: a selective historical review and progress report. In W.M. Cowan (Ed.), Studies in Developmental Neurobiology: Essays in Honor of Viktor Hamburger, Oxford University Press, New York, 1981, pp. 74-133. 32 Perry, V.H., Henderson, Z. and Linden, R., Postnatal changes in retinal ganglion cell and optic axon populations in the pigmented rat, J. Comp. Neurol., 219 (1983) 356-368. 33 Perry, V.H. and Linden, R., Evidence for dendritic competition in developing retina, Nature (Lond.), 297 (1982) 683. 34 Perry, V.H., Oehler, R. and Cowey, A., Retinal ganglion cells that project to the dorsal lateral geniculate nucleus in the macaque monkey, Neuroscience, 12 (1984) 1101-1123. 35 Peters, A., Palay, S.L. and Webster, H., The Fine Structure of the Nervous System: The Neurons and Supporting Cells, Saunders, Philadelphia, 1976, 406 p. 36 Potts, R.A., Dreher, B. and Bennett, M.R., The loss of ganglion cells in the developing retina of the rat, Dev. Brain Res., 3 (1982) 481-486. 37 Provis, J.M., van Driel, D., Billson, F.A. and Russell, P., Human fetal optic nerve: overproduction and elimination of retinal axons during development, J. Comp. Neurol., 238 (1985) 92-100. 38 Rakic, P. and Riley, K.P., Overproduction and elimination
48
39
40
41
42
43
44
45
of retinal axons in the fetal rhesus monkey, Science, 219 (1983) 1441-1444. Rakic, P. and Riley, K.P., Regulation of axon number in primate optic nerve by prenatal binocular competition, Nature (Lond.), 305 (1983) 135-137. Reese, B.F, The topography of expanded uncrossed retinal projection following neonatal enucleation of one eye: differing effects in dorsal lateral geniculate nucleus and superior colliculus, J. Comp. Neurol., 250 (1986) 8-32. Reynolds, H.C., Studies on reproduction in the opossum (Didelphis virginiana), Univ. Calif. Publ. Zool., 52 (1962) 223-275. Robinson, S.R., Horsburgh, G.M., Dreher, B. and McCall, MA., Changes in the number of retinal ganglion cells and optic nerve axons in the developing albino rabbit, Dev. Brain Res., 35 (1987) 161-174. Sefton, A.J. and Lam, K., Quantitative and morphological studies on developing optic axons in normal and enucleated albino rats, Exp. Brain Res., 57 (1984) 107-117. Sengelaub, D.R. and Finlay, B.L., Cell death in the mammalian visual system during normal development. I. Retinal ganglion cells, J. Comp. Neurol., 204 (1982) 311-317. Shatz, C.J. and Sretavan, D.W., Interactions between reti-
46
47
48
49
50
51
nal ganglion cells during the development of the mammalian visual system, Annu. Rev. Neurosci., 9 (1986) 171-207. Sretavan, D.W. and Shatz, C.J., Prenatal development of cat retino-geniculate arbors in the absence of binocular interactions, J. Neurosci., 6 (1986) 990=i003: Stone, J., Parallel Processing in the Visual System: The Classification of Retinal Ganglion Cells and its Impact on the Neurobiology of Vision, Plenum, New York, 1983, 438 p. Tay, D., So, K.-F., Jen, L.S, and Lau, K.C., The postnatal development of the optic nerve in hamsters: an electronmicroscopic study, Dev. Brain Res, 30 (1986) 268-273. Williams, R.W., Bastiani, M.J. and Chalupa, L.M., Loss of axons in the cat optic nerve following fetal unilateral enucleation: an electron microscopic analysis~ J. Neurosci., 3 (1983) 133-144. Williams, R.W., Bastiani, M.J., Lia. B. and Chalupa, L.M., Growth cones, dying axons, and developmental fluctuations in the fiber population of the cat's optic nerve, J. Comp. Neurol., 246 (1986) 32-69. Williams, R.W. and Rakic, P.R., Dispersion of growing axons within the optic nerve of the embryonic monkey, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3906-3910.
37
BRD 50825
Development of the optic nerve of the opossum
( Didelphis virginiana) Michael A. Kirby 1, Paul D. Wilson 2 and Thomas M. Fischer 2 IDepartments of Pediatrics and Anatomy, School of Medicine, Loma Linda University, Loma Linda, CA 92350 (U.S.A.) and 2Department of Psychology, University of California, Riverside, Riverside, CA 92521 (U.S.A.) (Accepted 5 July 1988) Key words: Development; Optic nerve; Enucleation; Re-organization
The development of the optic nerve of a marsupial, the North American opossum, was examined in 24 animals from postnatal days 5 to 78 (P5-P78); gestation is 13 days. The estimated number of axons increased from 24,000 at P5, to 267,000 at P27, approximately 2.7 times the mean number in the adult. Following P27, axon numbers decreased rapidly to 140,000 at P40, then decreased more slowly, attaining adult values between P50 and P59. Thus, the opossum is similar to placental mammals examined in evidencing an overproduction and later attenuation to adult values in the number of axons in the optic nerve during development. Monocular enucleation of 3 animals at P17, 10 days before peak axon counts, resulted in a mean population increase of 24,000 (range 19,000-30,000) above the normal adult mean. Additionally, a 4th animal monocularly enucleated on P7, 3 days prior to the arrival of migrating fibers to central target sites, had a similar value of 26,500 supernumerary axons. Our findings in the opposum, when coupled with previous reports in other mammals, suggest that binocular interactions during development account only for optic nerve axon loss approximately equal in magnitude to the ipsilateral projection from one eye.
INTRODUCTION Initial o v e r p r o d u c t i o n and subsequent reduction of axon n u m b e r s in the optic nerve during development have been o b s e r v e d in the rat t°'14As'25,32, hamster 48, rabbit 42, marsupial native cat 11 and q u o k k a 3 (although see ref. 1), m o n k e y 38, human 37 and cat 30'49, and have been c o r r e l a t e d with the overproduction and elimination of retinal ganglion cells in the rat 25'32,36, rabbit 42, h a m s t e r 44, and cat 7. While all the contributing mechanisms are not fully understood, it has been suggested that this p h e n o m e n o n , the substantial reduction in axon numbers, reflects competition for terminal synaptic space 7'12'31'49. The evidence that binocular competition is an i m p o r t a n t mechanism contributing to the reduction of axon numbers in the developing optic nerve derives principally from studies d e m o n s t r a t i n g that m o n o c u l a r enucleation
near the time when axon counts p e a k reduces the severity of axon loss in the remaining optic nerve. Counts of axons in the remaining nerve of mature animals monocularly enucleated during early develo p m e n t evidence little or no increase above normal in the rat 43, 25% increase in the cat 49, and 34.7% increase in the m o n k e y 39. A t present, the reasons for this species difference is not understood. Previous investigators 39'43 have n o t e d that the magnitude of the increase in axon n u m b e r s is related to the size of the ipsilateral population in each of these species. The rat has a small ipsilateral projection that averages less than 4 % of the total ganglion cell population in p i g m e n t e d animals 15,2° and less than 2% in albinos 15'43, c o m p a r e d to a p p r o x i m a t e l y 28% in the cat (ref. 47, p. 283) and 40% in the m o n k e y 34. The opossum has a substantial ipsilateral projection (approximately 20% of the ganglion cell population, see ref.
Correspondence: M.A. Kirby, Department of Pediatrics, School of Medicine, Loma Linda University, Loma Linda, CA 92350, U.S.A. 0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
38 23, Fig. 1; personal observations) and offers the opportunity to examine the magnitude of increase in the number of optic nerve axons following early monocular enucleation in an animal that has an ipsilateral projection intermediate in size between the rat and cat. Most studies which have examined the effects of disruption of binocular competition during development have removed one eye near the time when the number of axons has peaked. This opens the possibility of confounding the consequences of disruption of binocular competition with the effects of axon degeneration or denervation of central target sites 45. Monocular enucleation performed prior to the arrival of retinal afferents at central target sites provides an opportunity to distinguish the effects of binocular competition from the effects of denervation or degeneration at central targets. In the present study we have examined the normal development of the optic nerve and the effects of early monocular enucleation on axon counts in the remaining optic nerve of the mature animal enucleated either 10 or 20 days before optic nerve counts normally peak. As a marsupial, the opossum is an ideal species for the study of early neural development. Opossum pups are accessible in the pouch at a relatively young age (born following 13 days gestation, ref. 41), and remain in the pouch until their eyes open at about 60 days after birth. A partial report of these findings has been given 24. MATERIALS AND METHODS
Experimental animals Twenty-four pouch-young animals, obtained from 7 litters, were used in the present study. Postnatal ages were established from known birthdates (2 litters), or estimated (5 litters) by body weight and snout-rump lengths using the method of Cutts et al.~3. Known birthdates were established by checking daily the maternal marsupium for the presence of young, the appearance of which was counted as postnatal day I (P1). The ages obtained from these observations agreed closely with the calculated age using body measurements ~3.
Histologicalprocedures Following either i.p. injections of sodium pento-
barbital or hypothermia-induced anesthesia, animals were perfused through the heart with buffered phosphate (pH 7.6), followed by a mixture of 4% paraformaldehyde 2% glutaraldehyde in the same buffer. The optic nerves with the globe attached for orientation were then removed and stored at room temperature in the fixative for 12-24 h, a step found to be critical for the preservation of the uitrastructure of the individual axons. Care was taken during the removal of the tissue to minimize physical distortion and to prevent desiccation by bathing the tissue with buffered fixative during dissection. The tissue was then postfixed in 1-2% osmium, washed in several' changes of buffered phosphate, dehydrated through an ascending series of acetone, and embedded in Spurr's plastic. Within each block, the optic nerve was carefully oriented to insure that all sections saved for analysis were cut directly transverse to the long axis of the nerve. Since axons may extend through the nerve at different times and with varied rates of growth, the numerical data obtained at a particular age and the measures of changes across ages could be substantially affected if samples were taken at variable distances along the nerve. To minimize such problems, the ultrathin (grey to grey-silver) and semithin (gold to purple) sections were obtained from each nerve at 1.2-2.5 mm from the globe. Compression in the ultrathin sections was relieved by xylene vapor and in the semithin sections by mild heat. Ultrathin sections were mounted on copper mesh grids (#300), stained with uranyl acetate and lead citrate, and photographed on either a Hitachi H-500 or H-600 electron microscope. Contrast for the youngest tissue was improved by prolonged staining times (2 h) in the uranyl acetate.
Sampling procedure Critical to the study was the use of a sampling procedure that would uniformly cover each nerve crosssection. For each nerve, electron micrographs were taken within each grid opening containing neural tissue, and for most nerves, 4 adjacent but non-overlapping micrographs were taken within each grid opening. For the late postnatal nerves (>P50) the number of grid squares available was greater and only two micrographs in diagonal corners of each grid square were obtained. The areas photographed were chosen
39 without regard for the relative amounts of the region occupied by axons or glia, but with consistent reference to the grid bars. Large blood vessels were not photographed. This type of sampling procedure was shown earlier 22 to sample all regions of the nerve uniformly and is similar to the method of Williams et al. 49'5° (see ref. 50 for a discussion of the advantages of this sampling technique). Electron micrographs were taken at a magnification of approximately 5000x, and enlarged in printing to a final magnification of 15,000-16,000x. Care was taken to ensure consistent magnification through the use of a calibration grid (Pelco #606). For the first sets of nerves the calibration grid was photographed at the beginning and end of each session. Since in each case the two measurements agreed within 0.1%, the calibration grid was photographed only once for each filming session for the remaining nerves. The total number of neural processes (axons and growth cones) in each nerve was estimated by counting the number of processes in the areas sampled in the ultrathin section and extrapolating to the entire cross-sectional area of the nerve (minus the large vascular elements) measured from the adjacent semithin section. No correction for differential compression was used, given evidence in a previous study 22 and in the present material that area differences between adjacent ultrathin and semithin sections were consistently less than 4%, and typically 1-2%. A random sample of neural processes for area measurement was obtained by drawing a diagonal line across each micrograph and measuring all processes which came in contact with the line. Myelin sheath when present was excluded in the measurement. Measurements of the processes were made on a computer-linked digitizer (Houston Instruments Hipad), and the area of each process was computed using a morphometry program (Bioquant II, R & M Biometrics). Distributions and descriptive statistics were generated using an SAS statistical package. Monocular enucleation
Optic nerve counts were obtained from 4 animals monocularly enucleated at P17 or P7. The female (containing the pouch young) was anesthetized (40 mg/kg Nembutal, i.p.) and the marsupium gently
opened to permit access to the young. Following administration of topical anesthetic (benzocaine), theeyelids of each pup were gently parted and the globe rotated free of the socket and removed. The socket was then repeatedly flushed with antiseptic and anesthetic (iodine and benzyl chloride), and the marsupium closed. For each animal the anesthetic level appropriate for enucleation was sufficient to eliminate all reflexive movement in response to a forceps pinch to the eyelids and surrounding tissue, and was attained 2-3 min after anesthetic application. RESULTS Optic nerve processes
Figs. 1 and 2 illustrate examples of the micrographs taken from the optic nerve of animals at different ages. The axons of young opossums, like those of adult opossums z2, could be distinguished from surrounding glial elements by the presence of microtubules and neurofilaments, as well as by the relative translucence of the axonal cytoplasm. Even in the youngest animals examined (P5, P9 in Fig. 1) the translucent quality of the axoplasm contrasted sharply with the darker glial processes which contained numerous fine fibrils and ribosomes as described previously for the opossum z2 and other mammals 35,5°. Also present in the youngest nerves were relatively large, irregular processes that resembled the axons in their subcellular components, but were presumed to be growth cones on the basis of their similarity to processes identified in the cat and monkey5°,51. These processes occasionally contained numerous organelles (i.e. clear vesicles, microfilaments, endoplasmic reticulum) characteristic of the bulbous central core of neural growth cones 5°. The majority of these processes had few of these complex subcellular components and were composed principally of a mesh of fine microfilaments in a translucent cytoplasm, similar in appearance to the most distal portions of neural growth cones in the cat and monkey5°'51. In the present study, the profiles presumed to be growth cones clearly differed from glial processes by characteristics which distinguished axons from glia, and were identified in the youngest nerves examined (e.g. P9, P10 in Figs. 1 and 2). Occasionally axon profiles were encountered that contained either darkly mottled cytoplasm or an ir-
m (gestation in this species is 12.5 days). Pres,inr ...nrn t.m;nn,,.. I,“,., ;r
41 regular membrane, but were otherwise surrounded by axons that were normal in appearance. Such profiles are morphologically similar to necrotic axon profiles described in the developing optic nerve of the c a t 49 (see also ref. 9), rat 43, and monkey 38. These processes were observed in the youngest ages examined, suggesting that some axon loss from the putative nerve occurs very early, even before the majority of migrating neurons reach central target sites. Although these observations are consistent with findings in the fetal cat 5°, our inability to identify these processes conclusively as necrotic (e.g. with enzyme markers, pyknotic nuclei) prevented a quantitative analysis of their numbers. What is clear is that very few of these profiles could be identified at any stage of development, suggesting that axon loss (through cell death or retraction) is a rapid process not readily identified with routine electron microscope techniques.
Axon numbers Fig. 3 illustrates the calculated axon population in each optic nerve plotted as a function of postnatal age. At P5, the axon population was estimated at 24,000, approximately 25% the mean adult value of 100,000. Over the next 3 weeks the number of optic nerve fibers increased rapidly, reaching 250,000 by the end of the third postnatal week. Maximum counts were obtained in our series at P27, with an estimated optic nerve fiber population of 267,000, approximately 2.7 times the mean adult value. Over the next few days the number of axons decreased to approximately 240,000 by P31 and 140,000 by P40. Following P40, axon counts gradually decreased and reached adult values between P50 and P59. To investigate the reliability of our sampling procedure, both optic nerves were counted in 5 animals. At P30 this variability was 3.0% (252,875 vs 245,188) and 3.4% (239,124 vs 230,975). In the remaining ani~ mais, the oldest examined (Fig. 2), the variability between the two nerves ranged from 4.2% (106,993 vs 102,498) to only 0.4% (110,679 vs 110,273) and 0.3% (107,843 vs 107,482). Since variance between the two nerves is equal or less than that observed when the same adult nerve is sectioned in two different locations e2, we feel fairly confident that our sampling procedure provided a reasonable degree of reliability.
Disruption of binocular competition To examine the effects of binocular competition on the remaining optic nerve, 3 opossum pups were monocularly enucleated on P17, 10 days prior to peak axon counts in normal animals. Retinal fibers first invade the thalamus on P10 (refs. 5, 6, 29) in D. marsupialis aurita, a species closely related to and having the same gestation period (12.5 days) as D. virginiana. The possibility exists that enucleation at P17 may cause axonal degeneration and denervation within central target sites, which might affect the number of axons in the remaining nerve, thereby potentially confusing secondary effects of denervation and degeneration with binocular competition 45. To minimize this possibility, one additional animal was enucleated on P7, which is approximately 3 days prior to arrival of fibers at their central target sites in D. marsupialis aurita and 20 days prior to peak axon counts found in this study. Counts were obtained from the remaining optic nerve of these 4 animals between P75 and P78, at which time the fiber population in normal animals is within the adult range. The mean fiber population for the P7 enucleate (126,508) was not substantially different from the P17 enucleates (119,231; 122,000; 129,790). The mean population for the 4 enucleates was 124,382 compared to 103,854 in 5 age-matched controls (range 94,271-110,679; Fig. 2).
Axon size Although the presumed growth cones were often markedly larger in area than axons (Fig. 1), the distribution of the cross-sectional areas of the two were continuous, as has been observed in other species (e.g. refs. 43, 50). Rather than attempting to distinguish between growth cones and axons of similar cross-sectional area on the basis of subcellular components and morphology, we included growth cones in the measurement of axons. Fig. 2 illustrates the axons (and growth cones when present) from the optic nerve of opossums at various ages. Cross-sectional areas ranged 0.001-1.21 /~m2 from P5 to P18. At later ages cross-sectional areas diminished and were 0.002-0.53/~m 2 at P24 and P27. The loss of profiles greater than 0.6/~m 2 in area paralleled an apparent decrease in growth cones which were frequently observed from P5 to P10, but rarely encountered in micrographs at P24 and P27. Following P27, the range
42
43
P75
Adult Fig. 2. Electron micrographs from the optic nerve of opossum pups at postnatal ages 10-75 (P10-P75) and the adult. At early ages numerous profiles were present, presumed to be growth cones, suggesting the continuing addition of axons to the developing optic nerve. At P27, the age having peak counts in our material, few growth cone processes were encountered. By P59, individual axons were fewer in number overall, and had larger cross-sectional areas (large arrow denotes early myelinated process). At P75 the majority of profiles were myelinated (large arrow), although several unmyelinated processes were still present (small arrows). These were smaller in cross-sectional area and had relatively less myelination than encountered in the adult. Bar = 1/~m.
of axon sizes increased to 0.003-1.69 pm 2 in area at. P50 and 0.038-2.94pm 2 at P78. Axons in the remaining nerve of 4 enucleated animals at P75-P78 were 0.018-3.55 pm 2 in area, similar to those in normal animals. Although the total number of axons in the nerve at P59-P78 is within the range found in the adult, the largest axons found at P78 were still considerably smaller in diameter than those in the adult. In adult optic nerve 22, axons excluding myelin are 0.12-6.06 pm 2 in diameter (equivalent to 0.01-28.8 /~m2 in area, if profiles are round). Apparently the
major increase in axon caliber occurs after the period of axon loss and the attainment of adult numbers. Correlated with developmental increase in the diameter of axons was the onset of myelination. The first myelinated fibers were present in our material at P50 when axon numbers were near normal for the adult; less than 2.0% were myelinated at this age. Thereafter, the number of myelinated fibers began to increase rapidly; 11% of the fibers were myelinated by P59 and 34% by P78. This is consistent with the finding in other mammals that myelination of optic fi-
44 bers begins lized30,38.43
when
axon
numbers
have
stabi-
Growth of the optic nerve The size of the optic nerve increased progressively with postnatal age. At P9 the cross-sectional area was only 0.009 mm 2, less than 2.4% of the mean adult area of 0.38 mm 2 reported previously 22. Present in the nerve at early postnatal ages but absent in the adult were numerous septa that divided the optic fibers into discrete fascicles. The size of each fascicle was not correlated with its position within the nerve; fascicles containing large numbers of axons were just as numerous near the center of the nerve as towards the periphery. The number of fibers within each fascicle varied considerably; however, in general the number of processes within each fascicle initially increased at early ages until P27. At P5, individual fascicles contained only 20-40 fibers whereas by the end of the third postnatal week individual fascicles contained 100-200 fibers (Fig. 2). From the end of the third postnatal week on, the number of processes within each fascicle decreased until by P50 only 10-20 axons were present within each fascicle. By P59 no fasciculation was apparent, which is characteristic of the adult opossum. The total nerve crosssectional area at P50 was 0.071 m m 2 (19% of the adult size 22) and 0.11 mm 2 (30% of adult) at P78.
,300,000 t 250,000J m
0(D
O
200,000
z0 150,000 X <:(
1oo,00o
! 8
50,000 0 ' 1'0' 2'0' 3'0" 4'0" 5'0' 6'0" 7'0 8'0 POSTNATAL ACE (DAYS) Fig. 3. Plot of the estimated population of axons in the developing optic nerve of the opossum at different postnatal ages.
Filled circles denote 3 animals monocuIady enucleated during early postnatal development (see text).
DISCUSSION
Changes in axon number with development This study has described an initial increment and later attrition in the number of axons within the developing optic nerve of the North American opossum. Similar to the growing list of placental mammals, e.g. monkey 38. cat 3°'49. rabbit 42. hamster 48. rat 1°'25'32. human 37, the development of the opossum optic nerve exhibits successive phases of development. The first phase, probably starting near birth (at E12.5) and continuing to the end of the 4th postnatal week, is characterized by a rapid increase in the number of axons within the presumptive nerve. A peak value of approximately 270,000 axons. 2.7 times normal adult values, is attained by P27. This is followed by a second phase of relatively rapid axon loss. Bv P40 the number of axons has decreased to approximately 127,000, roughly 30,000 axons more than the average number in the adult. In the third phase, axon numbers decrease more slowly to reach normal adult values by about the 8th postnatal week. Thus axon numbers of this marsupial exhibit a development pattern quite similar to that seen in placental mammals. Technical considerations Consideration must be given to several technical problems that could potentially affect our observations. The first concerns the possible inclusion in our counts of multiple growth cone processes that originate from a single migrating fiber. In culture, the distal portions of axons divide into multiple filopodial extensions 4'8, and the use of a single cross-section through the developing nerve may result in an overestimation of the actual fiber population during periods where numerous growth cone processes are present. Recent observations by Mason (refs. 2, 27, and personal communication) indicate that the terminal morphology of growth cones is less complex within fiber pathways in vivo than within either target nuclei in vivo or preparations in vitro, suggesting that multiple growth cone processes may be less of a problem than would be predicted on the basis of the structural complexity observed in vitro. Furthermore, since the total number of axons increased almost linearly between P5 and Pt8 when apparent growth cone profiles were decreasing in number, the possible inclusion of multiple processes from the same cell proba-
45 bly contributed little to the estimate of the total axon population in this period. The developing optic nerve of the quokka has been reported to have axon numbers which greatly exceed the total estimated number of retinal ganglion cells and it has been suggested that optic axons may branch extensively in the quokka optic nerve during early development, with subsequent loss of collaterals later in development 3'16. However, the presence of branching profiles within the nerve may reflect fixation artifact, and the discrepancy between axon and ganglion cell counts may have resulted from the inability to label the entire ganglion cell population through retrograde transport techniques. We have succeeded in labeling 75% of the retinal ganglion cell population predicted from nerve axon counts in a P30 opossum (unpublished observations). Equivalent or higher correlation has been obtained between counts of nerve axons and cells in the ganglion cell layer during development in rat 32, rabbit 42, and cat (Lia and Chalupa, personal communication). Recently, Lia et al. 26, counting axons at the proximal and distal ends of the fetal optic nerve of the cat, found more axons near the eye than near the chiasm, arguing against significant bifurcation of axons within the nerve during development. A second potential problem concerns the method by which the total population of each nerve was estimated. We have shown previously in the adult 22, that sampling all grid squares containing neural tissue in a single cross-section reduces the chance for error arising from changing axon densities associated with axons of different diameters and glial processes. However, this method when employed in the relatively small nerves of the pouch-young opossum may introduce other problems (e.g. adequate sample area, coverage of neural processes by grid bars). To evaluate this possibility we analyzed both nerves in each of 5 normal animals. The difference in counts between the two nerves in these animals varied from 0.3 to 4.2% (Fig. 3). Thus we feel confident that this technique permits a relatively accurate assessment of the total population within the nerve of individual animals for both normal an enucleated animals. It is important to point out that the addition of axons to the developing nerve may be accompanied by simultaneous axon loss, although we could not identify with certainty necrotic axon profiles. The exis-
tence of numerous macrophages in the optic fiber layer of the retina as early as P22 (Kirby and Wilson, in preparation), several days before the sharp decline in axon numbers, supports the possibility that cell death begins well before the peak in retinal ganglion cell numbers has been obtained. Therefore, the actual number of fibers added to the developing nerve most likely exceeds the number reported here. Recently, Williams et al. 5° have calculated, from estimates of the number and time course of axon loss in the developing optic nerve of the cat, that approximately 20% of all axons are lostprior to the period of peak count. Taking into account the same ratio and time course for axon removal (1 h) used by Williams et al. 5° for the cat, the opossum optic nerve may have an actual peak fiber population as large as 324,000.
Development of retinal projections In close agreement with our findings on the development of the opossum optic nerve are the recent observations of the development of retinal projections to the superior colliculus of Didelphis marsupialis, a South American opossum 29. At P7 in this species, retinal fibers have reached only the rostral tip of the optic tract and the first retinal fibers arrive at the superior colliculus by P10. Retinal ingrowth to the superior colliculus continues to increase over the next 12 days, with maximum overlap between projections of the two eyes occurring on P22, a few days before the time of peak axon counts in the present study. Retraction of the exuberant ipsilateral projection to the adult pattern does not start until P26, which is approximately the time of the onset of greatest axon loss in the optic nerve in our material. The first laminar segregation of retinal terminals in the superior colliculus of D. marsupialis does not occur until P47, which is near the time period when axon counts in the optic nerve of D. virginiana attain adult values. Thus the time of ingrowth, expansion, and later retraction to the adult pattern of retinal afferents to the superior colliculus corresponds closely with our observed time-course of axon addition and later attrition in the optic nerve.
Effects of early monocular enucleation We have shown that early monocular enucleation in the opossum results in an increase above normal of approximately 24,000 axons in the remaining optic
46 nerve. This value corresponds to the number of ganglion cells estimated to project ipsilaterally in this species (unpublished observations) and suggests that elimination of binocular competition reduces cell death proportionate to the size of the ipsilateral projection. In the cat, which has an ipsilateral projection involving approximately 28% of the adult ganglion cell population 47, prenatal monocular enucleation increases the number of axons in the remaining optic nerve by 25% (ref. 49) and the number of ganglion cells in the remaining retina 7 of the adult. In the primate 39, which has 40% of the ganglion cell population projecting ipsilaterally34, monocular enucleation in the fetus increases the number of axons in the remaining nerve of the adult by 32-38%. In contrast to these two species, the size of the ipsilateral projection in the albino rat 15'43 is only 1% (approximately 1200 axons). Monocular enucleation during early development in this species does not result in an apparent increase in the number of axons in the remaining nerve of the adult25; however, normal interanimal variation in optic nerve numbers could presumably obscure any supernumerary axons that might result. The possibility that supernumerary axons may be present is suggested by observations that early enucleation results in greater than normal numbers of ganglion cells that project ipsilaterally in the remaining retina (e.g. refs. 17, 19, 20, 40). In each of these species, however, enucleation was performed at a time when ocular inputs from the two eyes had already reached central sites of termination, opening the possibility that factors other than binocular competition, e.g. re-routing at the chiasm, axonal degeneration, or relay cell denervation 19'45 might alter the numbers of fibers in the remaining nerve. To explore this possibility, we monocularly enucleated one animal at P7, prior to the ingrowth of retinal projections to central sites of termination. The number of axons in the remaining nerve of this animal did not differ significantly from the axon numbers in animals enucleated at P17. This is consistent with reports on
REFERENCES 1 Beazley, L.D. and Dunlap, S.A., The evolution of an area centralis and visual streak in the marsupial Setonix brachyurus, J. Comp. Neurol., 216 (1983) 211-231. 2 Bovolenta, P. and Mason, C., Growth cone morphology
the fetal cat, in which monocular enucteation during either early fetal periods (E23, refs. 45, 46) or relatively late 49 produces equivalent axon populations in the remaining nerve. Interestingly, in the cat 4s and rat ~9monocular enucleation at relatively early stages of retinal development (i.e. prior to the arrival of axons at the optic chiasm) results in a smaller than normal ipsilateral projection, suggesting that in addition to eliminating competition at central target sites, monocular enucleation may affect guidance of afferent fibers within the optic pathway, However, most findings to date imply that factors intrinsic to binocular competition at central target sites determine the magnitude of the supernumerary population, although other factors may also be important 21'33'45. Although speculative, this suggests that one effect of monocular enucleation is to increase the availability of terminal synaptic sites (or trophic factors) within the portions of retinorecipient structures having binocular representations, thereby maintaining cells that are otherwise lost through binocular competition. Some support for this is found in a recent observation that the majority (in absolute numbers) of supernumerary ganglion cells resulting from early monocular enucleation in the cat are located in the area centralis 7'21, where interactions during development between binocular competition and cell density would be maximum. The substantial body of evidence in the rat, mouse, hamster, and cat demonstrating the maintenance of an enlarged ipsilateral pathway in the mature animal following monocular enucleation during development would indicate that the principal effect of binocular competition is the retraction of retinal afferents coming from the inappropriate eye. It would be of interest to analyze ganglion cell receptive field properties and topographic organization (retinal location and number of ganglion cells) of central projection patterns for the remaining eye of adult animals monocularly enucleated during development.
varies with position in the developingmouse visual pathway from retina to first targets, J. Neurosci., 7 (I987) 1447- 1460. 3 Braekevelt, C.R., Beazley, L.D., Dunlap, S.A. and Darby, J.D., Number of axons in the optic nerve and retinal ganglion cells during development in the marsupial Setonix
47 brachyurus, Dev. Brain Res., 25 (1986) 117-125. 4 Bray, D. and Chapman, K., Analysis of microspike movements on the neuronal growth cone, J. Neurosci., 5 (1985) 3204-3213. 5 Cavalcante, L.A. and Rocha-Miranda, C.E., Postnatal development of retinogeniculate, retinopretectal, and retinotectal projections in the opossum, Brain Res., 146 (1978) 231-348. 6 Cavalcante, L.A., Rocha-Miranda, C.E. and Linden, R., Observations on postnatal neurogenesis in the superior colliculus and the pretectum in the opossum, Dev. Brain Res., 13 (1984) 241-249. 7 Chalupa, L.M., Williams, R.W. and Henderson, Z., Binocular interaction in the fetal cat regulates the size of the ganglion cell population, Neuroscience, 12 (1984) 1139-1146. 8 Connolly, J.L., Seeley, P.J. and Greene, L.A., Regulation of growth cone morphology by nerve growth factor: a comparative study by scanning electron microscopy, J. Neurosci., 13 (1985) 183-198. 9 Cooke, R.D., Ghetti, B. and Wisniewski, H.M., The pattern of Wallerian degeneration in the optic nerve of newborn kittens: an ultrastructural study, Brain Res., 75 (1974) 261-275. 10 Crespo, D.D., O'Leary, D.M. and Cowan, W.M., Changes in the numbers of optic nerve fibers during late prenatal and postnatal development in the albino rat, Dev. Brain Res., 19 (1985) 129-134. 11 Crewther, D.P., Nelson, J.E. and Crewther, S.G., Afferent input for target survival in marsupial visual development, Neurosci. Lett., 86 (1988) 147-154. 12 Cunningham, T.J., Mohler, I.M. and Giordana, D.L., Naturally occurring neuron death in ganglion cell layer of the neonatal rat: morphology and evidence for regional correspondence with neuron death in superior colliculus, Dev. Brain Res., 2 (1982) 203-215. 13 Cutts, J.H., Kruse, W.J. and Leeson, C.R., General observations on the growth and development of the young pouch opossum, Didelphis virginiana, Biol. Neonate, 33 (1978) 264-272. 14 Dreher, B., Potts, R.A. and Bennett, M.R., Evidence that the early postnatal reduction in the number of rat retinal ganglion cells is due to a wave of ganglion cell death, Neurosci. Left., 36 (1983) 255-260. 15 Dreher, B., Potts, R.A., Ni, S.Y.K. and Bennett, M.R., The development of heterogeneities in distribution and soma sizes of rat retinal ganglion cells is due to a wave of ganglion cell death. In J. Stone, B. Dreher and D. Rapaport (Eds.), Development of the Visual Pathways in Mammals, Liss, New York, 1984, pp. 39-57. 16 Dunlop, S.A. and Beazley, L.D., A retino-retinal projection contributes minimally towards high axon numbers in developing optic nerve of the marsupial Setonix brachyurus, Soc. Neurosci. Abstr., 12 (1986) 121. 17 Fawcett, J.W., O'Leary, D.D.M. and Cowan, W.M., Activity and control of ganglion cell death in the rat retina, Proc. Natl. Acad. Sci. U.S.A., 81 (1984)5589-5593. 18 Foster, R.E., Conners, B.W. and Waxman, S.G., Rat optic nerve: electrophysiological, pharmacological, and anatomical studies during development, Dev. Brain Res., 3 (1982) 371-386. 19 Godemont, P., SalaiJn, J. and M6tin, C., Fate of uncrossed retinal projections following early or late prenatal monocular enucleation in the mouse, J. Comp. Neurol., 255 (1987)
97-109. 20 Jeffery, G., Retinal ganglion cell death and terminal field retraction in the developing rodent visual system, Dev. Brain Res., 13 (1984) 81-96. 21 Kirby, M.A. and Chalupa, L.M., Retinal crowding alters the morphology of alpha ganglion cells, J. Comp. Neurol., 251 (1986) 532-541. 22 Kirby, M.A., Clift-Forsberg, L., Wilson, P.D. and Rapisardi, S.C., Quantitative analysis of the optic nerve of the North American opossum (Didelphis virginiana): an electron microscopic study, J. Comp. Neurol., 211 (1982) 318-327. 23 Kirby, M.A. and Wilson, P.D., Receptive field properties and latency of cells in the lateral geniculate nucleus of the North American opossum (Did@his virginiana), J. Neurophysiol., 56 (1986) 907-933. 24 Kirby, M.A. and Wilson, P.D., Axon count in the developing optic nerve of the North American opossum: overproduction and elimination, Soc. Neurosci. Abstr., 10 (1984) 467. 25 Lam, K., Sefton, J. and Bennet, M.R., Loss of axons from the optic nerve of the rat during early postnatal development, Dev. Brain Res., 3 (1982) 487-491. 26 Lia, B., Williams, R.W. and Chalupa, L.M., Overproduction of optic nerve fibers in the fetal cat does not reflect axon branching within the nerve, Suppl. Invest. Ophthaltool. Vis. Sci., 26 (1985) 286. 27 Mason, C.A., Growing tips of embryonic cerebeUar axons in vivo, J. Neurosci. Meth., 13 (1985) 55-73. 28 Mason, C.A., How do growth cones grow?, Trends Neurosci., 8 (1985) 304-306. 29 M6ndez-Otero, R., Cavalcante, L.A., Rocha-Miranda, C.E., Bernardes, R.F. and Barrados, P.C,R., Growth and restriction of the ipsilateral retinocular projection in the opossum, Dev. Brain Res., 18 (1985) 199-210. 30 Ng, A.Y.K. and Stone, J., The optic nerve of the cat: appearance and loss of axons during normal development, Dev. Brain Res., 5 (1982) 263-271. 31 Oppenheim, R.W., Neuronal cell death and some related regressive phenomena during neurogenesis: a selective historical review and progress report. In W.M. Cowan (Ed.), Studies in Developmental Neurobiology: Essays in Honor of Viktor Hamburger, Oxford University Press, New York, 1981, pp. 74-133. 32 Perry, V.H., Henderson, Z. and Linden, R., Postnatal changes in retinal ganglion cell and optic axon populations in the pigmented rat, J. Comp. Neurol., 219 (1983) 356-368. 33 Perry, V.H. and Linden, R., Evidence for dendritic competition in developing retina, Nature (Lond.), 297 (1982) 683. 34 Perry, V.H., Oehler, R. and Cowey, A., Retinal ganglion cells that project to the dorsal lateral geniculate nucleus in the macaque monkey, Neuroscience, 12 (1984) 1101-1123. 35 Peters, A., Palay, S.L. and Webster, H., The Fine Structure of the Nervous System: The Neurons and Supporting Cells, Saunders, Philadelphia, 1976, 406 p. 36 Potts, R.A., Dreher, B. and Bennett, M.R., The loss of ganglion cells in the developing retina of the rat, Dev. Brain Res., 3 (1982) 481-486. 37 Provis, J.M., van Driel, D., Billson, F.A. and Russell, P., Human fetal optic nerve: overproduction and elimination of retinal axons during development, J. Comp. Neurol., 238 (1985) 92-100. 38 Rakic, P. and Riley, K.P., Overproduction and elimination
48
39
40
41
42
43
44
45
of retinal axons in the fetal rhesus monkey, Science, 219 (1983) 1441-1444. Rakic, P. and Riley, K.P., Regulation of axon number in primate optic nerve by prenatal binocular competition, Nature (Lond.), 305 (1983) 135-137. Reese, B.F, The topography of expanded uncrossed retinal projection following neonatal enucleation of one eye: differing effects in dorsal lateral geniculate nucleus and superior colliculus, J. Comp. Neurol., 250 (1986) 8-32. Reynolds, H.C., Studies on reproduction in the opossum (Didelphis virginiana), Univ. Calif. Publ. Zool., 52 (1962) 223-275. Robinson, S.R., Horsburgh, G.M., Dreher, B. and McCall, MA., Changes in the number of retinal ganglion cells and optic nerve axons in the developing albino rabbit, Dev. Brain Res., 35 (1987) 161-174. Sefton, A.J. and Lam, K., Quantitative and morphological studies on developing optic axons in normal and enucleated albino rats, Exp. Brain Res., 57 (1984) 107-117. Sengelaub, D.R. and Finlay, B.L., Cell death in the mammalian visual system during normal development. I. Retinal ganglion cells, J. Comp. Neurol., 204 (1982) 311-317. Shatz, C.J. and Sretavan, D.W., Interactions between reti-
46
47
48
49
50
51
nal ganglion cells during the development of the mammalian visual system, Annu. Rev. Neurosci., 9 (1986) 171-207. Sretavan, D.W. and Shatz, C.J., Prenatal development of cat retino-geniculate arbors in the absence of binocular interactions, J. Neurosci., 6 (1986) 990=i003: Stone, J., Parallel Processing in the Visual System: The Classification of Retinal Ganglion Cells and its Impact on the Neurobiology of Vision, Plenum, New York, 1983, 438 p. Tay, D., So, K.-F., Jen, L.S, and Lau, K.C., The postnatal development of the optic nerve in hamsters: an electronmicroscopic study, Dev. Brain Res, 30 (1986) 268-273. Williams, R.W., Bastiani, M.J. and Chalupa, L.M., Loss of axons in the cat optic nerve following fetal unilateral enucleation: an electron microscopic analysis~ J. Neurosci., 3 (1983) 133-144. Williams, R.W., Bastiani, M.J., Lia. B. and Chalupa, L.M., Growth cones, dying axons, and developmental fluctuations in the fiber population of the cat's optic nerve, J. Comp. Neurol., 246 (1986) 32-69. Williams, R.W. and Rakic, P.R., Dispersion of growing axons within the optic nerve of the embryonic monkey, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 3906-3910.