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The position of the crossed and uncrossed optic axons, and the non-optic axons, in the optic tract of the rat
0306-4522/87$3.00+ 0.00 Pergamon Journals Ltd 0 1987IBRO
Neurmchce Vol. 22, No. 3, pp. 1025-1039, 1987 Printed in Great Britain
THE POSITION OF THE CROSSED AND UNCROSSED OPTIC AXONS, AND THE NON-OPTIC AXONS, IN THE OPTIC TRACT OF THE RAT B. E. b@SE University of Oxford, Department
of Human Anatomy, South Parks Road, Oxford OX1 3QX, U.K.
Abstrad-The position of the crossed and uncrossed optic axons, and of the non-optic axons, within the optic tract was determined in the adult hooded rat. Horseradish peroxidase bistochemistry and lesion-induced degeneration of axonal profiles were independently used to study the position of the three relevant populations of axons witbin the optic tract. The boundaries of the optic tract are distinct at all but its caudomedial border, where it abuts the supra-optic commissures running parallel to the fibres of the optic tract. Labelling the crossed population of optic axons, or inducing their degeneration, both demonstrate a clear caudomedial border of the optic tract, although a number of optic axons stray out of the optic tract and course within the supra-optic commissures immediately caudomedial to the tract. The uncrossed optic axons are, as a population, positioned relatively deep in the optic tract, towards its dorsal border. A few occur at further ventral positions, but their density is greatly reduced there. Tbere is also a very thin region along the dorsalmost edge of the optic tract free of uncrossed optic axons. The relative position of the uncrossed to the crossed optic axons is discussed in the context of the mammalian optic tractas a chronologicalmap:spatial position in the tract may reflect temporal order of axonal arrival during early development. A large population of non-optic axons belonging to Gudden’s commissure courses within tbe boundaries of the optic tract at a relitively ventral position. They are most frequent caudomedially, and are absent rostrolatcrally. Hence, axons of the optic tract and Gudden’s commissure are substantially intermingled in the caudomedial half of tbe optic tract. These non-optic axons greatly outnumber the uncrossed optic axons, and will consequently distort counts of uncrossed optic axons based on intact pro&s that remain after removal of the opposite eye. However, they are still a minority in comparison to the crossed optic axons in this region.
There is increasing evidence to demonstrate that the mammalian optic tract can he viewed as a chronological map of axonal arrival, as has been known for various non-mammalian species for some time.6**~L1~1z1g~27 In the cat, for example, there is a correlation between the temporal pattern of genesis of the different ganglion cell types with the position of axon diameter classes across the deep-to-superficial axis of the optic tract.gJ’ls In the ferret, if the
retinofugal projections are labelled during the period of ganglion cell genesis, they are observed to occupy the whole optic tract when examined 24 h later; yet the labelled fibres occupy only deeper portions of the tract if examined 4 days later, when the tract is much larger in size due to the addition of newly arrived fibres arising from recently generated ganglion cells.” And in the fetal mouse, profdes of axonal growth cones are found in the optic tract only near its outer border, subjacent to the superficial (pial) surface.’ These various results strengthen the belief that one may read temporal order out of spatial position of the optic fibres within the mammalian optic tract.
The present investigations examined the position of the uncrossed optic axons within the optic tract of the adult rat. The ipsilaterally projecting retinal ganglion cells in the rat represent less than 5% of the total retinal ganglion cell population, comprising about 20% of the ganglion cells situated within the temporal retina. 3~15Given that this population is so small relative to the contralaterally projecting retinal ganglion cells, it should be possible to identify the position of these uncrossed optic axons relative to the complete crossed projection and, from this, to infer their relative arrival times in the optic tract during early development. The present investigation also examined the position of the non-optic axons that course amongst the optic axons within the optic tract. They comprise a non-trivial proportion of axons with respect to the population of uncrossed optic axons, and their presence is likely to account for the substantially elevated estimates of the uncrossed population when based on counts of intact axonal profiles in the optic tract following contralateral eye removal.28 EXPERIMENTAL
Abbreuiations: dLGN, dorsal lateral geniculate nucleus; HRP, horseradish peroxidaq SC, superior colliculus; TMB, tetramethylbcnzidine.
PROCEDURES
Experiments employing horseradish peroxidase hirtochemflry to reveal axonal populations Seven adult male and female Long-Evans hooded rats 1025
1026
B. E. RnsE
were used. Surgical procedures for the horseradish peroxidase (HRP) injections into the eye and along the optic tract were identical to those described previou~ly.~‘~~Briefly, each rat was anaesthetited and placed in a stereotaxic headholder. They received either a unilateral eye injection (5~1 of a 40% HRP solution dissolved in 2% dimethyl sulphoxide) or a series of six stereotaxicaliy placed intracerebral injections (0.15 ~1 of same, per injection), the coordinates of which had been previously determined to locate the optic tract. Following either a 24 or a 48 h survival, respectively, rats were heavily anaesthetized and then transcardially perfused with 5Oml of 0.9% saline followed by 300 ml of 1.25% pamformaldehyde plus 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2 at 2OT). For those rats that received mtracerebral mjections of HRP. they were additionallv net-fused with lOOm1 of 1.O% pa&fo&raldehyde in b&r *(pH 7.2 at 4°C) immediately following the saline, and their eyes were then removed, after which the perfusion was continued with the paraformaldehy~ghnaraldehyde solution. Brains were removed and left to sink overnight in 2O% sucrose dissolved in buffer (4°C). Retinal fiatmounts were prepared and reacted for HRP histochemistry, as bef0re.u Brains were cut either coronally or horizontally at 5Opm on a freezing microtome. Sections were reacted for enzymatic activity using tetrametbylbcnxidine (TMB) or both TMB and pphenyknediamine plus catechol (alternate sections) as the chromogen(s) toXs Sections through the optic tract were. drawn with the aid of a drawing tube attached to the microscope at a final magnification of x 50. Experimen:s profles
to show the distribution o/&generaring
axonal
Six adult mak and femak Long-Evans hooded rats were used.Eachmtwasanaen&etizsdandpkcedinastereotaxic headholder. They received either a tmikteral emtckation or a surgical ablation of one optic tract. For the enuckations, the globe was removed in its entirety, the orbit Packed with gelfoam, and the eyelids sutured. For the optic tract transections, the Jcplp was opened, the doraolateral skull was removed, and an aspirative lesion was made under v&al control with the aid of an operating microscope. The overlying cortex and hippoeampLIs were at&ted to reveal the donolateral aspect of the optic tract. This was then aspiratedtoremovetheentirelateralgcnkukten~andto sever the entirety of the optic tract. The cavity was then
filkdwithgelfoamandthesealpsutured.Allratswerekft to survive for 9 or 10 days. They were then perfused. and their optic nerves and tracts were pmcemed for hgbt and ekctron microscopy. using procedures identical to those described in the pmceding pa~er,~ except that (I) only the intmcranialopticneNeawereprouamd,and(2)tbehamispbem containing the ablated optic tract was pkeed into 30% sucrose in 10% formalin, cut coronnBy at 5Opm on a freezing microtome, and a l-in-5 series was mounted and stained with Cmsyl Viokt acetate. Se&thin sections of the optic tract ipailateml to an enucknted aye or opposite to an abkteddomokteraltbakmuswemdrawnat x4tlOwiththe aidofadrawingtubeattachedtothemicroscope.andthe position of wte profike was inttiatted. All figurea and photomkrogmpha of uprimDnd dcgenarationandofintactaltmlttlpfol!&ailbWatamamtial inapkneorthogonaltothedimdiondthean0ea,and conaequantly do not cortwpond to any amndard berso4utc plane.Themmabdng6gttmsandpho~~~ 1, 7,8and9)showsectionscutinthecoronaipkne. In the optic tract contmlateral to the ettuckation of one rat, axon dknmtem were measured ffom &CtrCm micrographs for evuy intact axon contaisbad within a @on 543gm’, and from tbeae WtaaxonW distributions were conatmcted for a mmtber of loatb~ across the optic tract and supra-@c commksuru. These
procedures were identical to those described in the preceding paper.2’ RESULTS
The crossed and uncrossed
optic
axons
in the optic
tract
The intraocular injections of HIW produced homogeneous filling of the target visual nuclei, in particular the dorsal lateral geaiculate nucleus (dLGN) and superior colliculus (SC), with no evidence for a failure to label a portion of the retina hecause of insmiicient spread of the tracer in the eye or because of damage to the axonal bundles in the optic fibre layer. Figure 1 shows photomicrographs and line drawings of coronal saxions through the optic tract contralateral and ipsilateral to an eye injected with HRP. Contralaterally. the labcUing is darw and even throughout the optic tract (Fig. lb and d). Along the medial edge of the tract there is a region of white matter devoid of all but a few lab&d axons and, medial to this unlabelled region+ there is a separate population of labelled axons. The latter, labelled, axons form the accessory optic tract, coursing along the ventral surface of the brain to the medial terminal nucleus of the accessory optic system. The unlabelled axons between the accessory and main optic tracts contain the various supra-optic commiaaures and other non-commissural fibre pthways.n In the ipsilateral optic tract, labelled optic axons are found relatively deep in the optic tract, nearer its dorsal border (Fig. la and c). The labelled axons occupy this deep position throughout the length of the tract, beginning dorsally just behind the optic chiasm, and continuing in a dorsomedial position as the optic tract courses doraolaterally over the diencephalon. In coronal sections, there is a small region free of uncrossed axons at the very dorsal and lateral extremes of the tract, along the tract’s deep border (Fig. la and c). The distribution of the crossed and uncrossed optic axons was largely confirmed by the location of degenerate profiles observed in the optic tracts ipsilateral and contralateral to an enu&ation. Figure 2 is a photomicrograph of the optic tract ContU to the enucleation. Degenerate pro&s are okserved caudomedially in the accessory optic tract (arrowheads); rostrolateral to this, intact axonal pr06ks Further roatroform the supra-optic corm&axes. laterally, the optic tract is dktinct at all but its caudomedial boundaries, where Gwkbed’s cammissure runs adjacent to the. optic axons. A few optic axons stray out of the region of &rMy pIcked degenerate pro&s, but a rough border can still be discerned (broken tine in Fig. 2). In the tract ipsilateral to the enuclation (Fig. 3), degenerate pro&s are found ptimarily deep in the tract, removed from the pial surface. Ssms i&Meral axons can be found at relatively soperiichl krcations
Fig. 1. Photomicrographs [(a} and fb)] and line drawings [(c) and (d)] of coronal sections through the optic tract ipsiiateral [(a) and (c)] and contralateral [@) and (d)] to a unilateral intra-oculax injection of HRP. Spacing between the sxxtions in (c) and (d) is 400 pm, with rostral sections to the top. Dorsal is up, medial is to the centre. (a), (b) 86-38. Bar = 100 pm. (c), (d) 86-37.
b
dorsal
caudo-medial
J
Fig. 2. 1028
opticand non-opuc axons in the optic tract (Fig. 3b), but the vast majority are nearer to the tract’s dorsal border. The degenerate profiles secll dorsally tend to be conspicuously larger than those observed ventrally, as might be expected from the axon diameter sixes described in these two regions of the tract in the preceding article.u very few degenerate pro&s are found immediately adjacent to the tract’s dorsal border (Fig. 3a and b). It was expected that this pattern of degeneration in the tract ipsilateral to enucleation (Fig. 3) would be detected by the complementary pattern of intact axons seen in the tract contralateral to enucleation (Fig. 2). Indeed, many intact large and small diameter axons were observed in this very dorsal region of the contralateral optic tract (Fig. 2a). But in addition there were a surprising number of intact axonal profiles, most of a smaller diameter, found primarily caudomedially in the tract (Fig. 2b). Figure 4 displays electron micrographs from three regions of an optic tract contralateral to an enucleation. Dorsally, deep in the optic tract, both some large and small diameter axons, though relatively few in number, appear intact (Fig. 4a), while caudomedially many more intact profiles ate present, though of smaller diameter (Fig. 4b). Rostrolaterally, very few unambiguously intact profiles are observed (Fig. 4~). Figure 5 displays the distribution of the axon diameters of these intact profiles at various locations across the optic tract contralateral to an enucleation, for every axon contained within regions 543 pm*. Large and small axons are found in that region of the tract known to contain degenerating profiles when the ipsilateral eye is removed (Fig. 5, histograms l-4; see Fig. 3), the coarser axons comprising a much greater proportion of the total than seen in the population of crossed, or crossed plus uncrossed, optic axons @receding pape?‘). Yet the number of intact axons here is insignificant in comparison with the large number of intact axons of small diameter (2 pm or less) in the caudomedial region of the optic tract (histograms 10-14, 17 and 18). Here, the definition of an intact axonal profile is based on the absence of those characteristics typical of degenerating axons: darkening of the axoplasm, disintegration of axoplasmic organdies, empty regions within the axon, and/or a disruption or shredding of the myelin lamellae. These features are rarely present in material examined from normal, nonexperimental, rats. The presence of these features is dependent upon survival time and on the diameter of the axon in question.M Hence, the failure to detect
1029
such protbes may not be a reliable indication that a given axon did not arise from the enucieated eye. However, this seems an unlikely explanation for the preset~e of these intact axons, since the axon diameter distribution in this region of the tract (caudomedUy) does not dilfer from locations rostrolaterally (see preceding article), where virtually all axonal profiles are degenerate following enucleation (Fig. 2c, 4c, Fig. 5, histograms 6-9, 15 and 16). Furthermore, few unambiguously intact axons can be found in the optic nerve of the enucleated eye ten days following enucleation (Fig. 6). For these reasons, it seems likely that these intact axons in the tract do not come from the contralateral retina. These same reasons make it unlikely that the intact axonal pro&s seen caudomedially arise from the ipsilateral retina but fail to transport significant amounts of HRP and do not show a degenerative response 10 days following enucleation. Nevertheless, the following experiments were undertaken to demonstrate that there is a population of commissural, non-optic, axons in the same region of the optic tract that contains these intact axonal profiles subsequent to removal of the contralateral eye. The non-optic axons in the optic tract Figure 7 shows a typical injection of HRP into the optic tract and dorsolateral thalamus. The injections of HRP along one optic tract produced heavy labelhng of the contralateral and ipsilateral retinal ganglion cell populations. The complete contralateral retina was labelled, as was the ipsilateral retina’s temporal crescent, and many of the cells displayed a Golgi-like labelhng indicative of uptake of the tracer by axons that were damaged by the syringe needle. These injections of HRP consistently labelled axonal processes in the contralateral optic tract. Figure 8a is a photomicrograph displaying label in the opposite optic tract typical of the present series of rats. In coronal sections, the labeiled axons occupy a ventral, pial position, and can be followed caudally in a progressively dorsomedial position near the tract’s caudomedial border (Fig. 8b). Although this population of labelled axons of the supra-optic conunissures cannot be readily defined with respect to the caudomedial border of the optic tract in this material, by comparing these results (Fig. 8) with those showing the crossed optic axons (Fig. l), it is clear that a large majority of these labelled profiles are well within the limits of the “optic” tract. This regional localization of non-optic axons
Fig. 2. Semithin section through the optic tract contralateral to an cnuckation ten days previously. Note the intact axonal profiles doraally (a), and aho~au~Iomedially (b), within the optic tract. Also note the &generate prohles at the far kft of the photormaogroph, in the accessory optic tract (arrowbatds). The broken line indicati the caudomedialborderof the optic tmct. (a)-&) Detaila from the re@onrindicated in the main photogrpph,dorsally. caudo~y and mstrokterally, mspcetively,from a nearbytion. 86341. p-I%enylcncdiaminestain. Bar = 50 pm for the main photorpaphand 25 pm for (a)-(c).
T
Fig. 4. Electron micrographs from the optic tract contralateral to an enucleation ten days previously. (a)+) Dorsal, caudomedial and rostrolateral optic tract, respectively, as in Fig. 2. 86-341. x6100. 1031
1032
B.E. REF~
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E~ _= ..=
8~
~8.o E~
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Optic and non-optic axons in the optic tract
Fig. 4. Electron micrograpb of an optic nerve ipsiiatersf to an enucleationten days previously.Not6 that virtually all of the axonal proNes appear degenerate. 86412L. x 6100.
within the optic tract was confirmed by the present of degenerate profiles following ablation of the opposite optic tract. Figure 9 displays a coronal section typical of the damage produced in two rats receiving ablations of the optic tract. At this coronal level, rostrai to the lateral genicuiate nuclei, the extent of damage to the dorsolateral thalamus can be seen, which extends caudally to the medial geniculate
nucIeus. The optic tract in this section (arrowhead in Fig. 9) is heavily gliotic, being completely transected further caudally. Figure 10 shows the pattern of degenerate axonal profiles following ablation of the opposite optic tract. The degenerate axons are positioned ventrally and caudomedially, both within the optic tract, and in the supra-optic commissures. Degenerate profiles are ab-
Fig. 7. Photomicrograph of a coronalsa%iondis&ying n typical injection a&eof heish pemxidase into the optic tractand dorsokitcral thalamus in the pnsent series of rats. 84-552.CresyI Violet acetate counterstain. Bar = I mm.
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b
Ontic and non-optic axons in the optic tract
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Fig, 9. Photomicrograph of a coronal section through the hemisphere that had been given an aspirative lesion of the dorsolateral thalamus. The arrowhead indicates the optic tract, in this section rostra1 to its transection. 86-237. Cresyl Violet acetate stain. Bar = 1mm.
sent from the deep, dorsal, part of the optic tract, where the majority of uncrossed axons course, and
from the rostrolateral optic tract, where no intact axonal profiles are observed following contralateral eye removal. Although there are many degenerate profiles in the caudomedial optic tract, Figs 2 and 10 indicate that the vast majority of axons in this region arise from the contralateral retina. Degenerate profiles in Figs 3 and 10 together, then, account for the distribution of intact axons seen in the optic tract after enucleation of the contralateral eye (Fig. 2). DISCUSSION
The present study has
The position of the uncrossed optic axons In the cat and monkey, the distributions of the crossed and uncrossed optic axons in the optic tract are almost completely overlapping, nearly filling the full extent of the optic tract’s boundaries. The exception in the cat concerns a small pure crossed portion of the tract, situated dorsolaterally, in that part of the optic tract representing inferior retina.” This discrepancy between the domains of the crossed and uncrossed optic axons has been interpreted in terms of the dissimilar periods of ganglion cell genesis
b
c Fig. 10. 1036
Optic and non-optic axons in the optic tract
for cells in the inferior nasal (contralaterally projecting) and inferior temporal (ipsilaterally projecting) retina. The absence of this pure crossed region ventromedially (the other extreme along the deep border of the cat’s optic tract), where the tract represents superior retina, corresponds well with the less discrepant periods of retinal ganglion cell genesis in superior nasal and superior temporal retina.9 In the rat, the present results describing the position of the uncrossed optic axons, arising from a proportionately smaller number of ipsilaterally projecting retinal ganglion cells, may be interpreted in the context of the optic tract as a chronological map (see introductory paragraphs). Although the relevant birthdating studies have not been done in the rat, studies of ganglion cell genesis in the mouse may have sutlicient generality for the temporal order of events in ratsSJg Ipsilaterally and contralaterally projecting retinal ganglion cells in the mouse are generated between days E-l 1 and E-17, but the contralaterally projecting cells continue to be generated until E-19.’ The peak production period for the ipsilaterally projecting cells is around E-13114.’ One might expect then that the bulk of the uncrossed optic axons would tend to occupy a relatively deep position in the optic tract, and that there would be few, if any, uncrossed optic axons along the pial surface of the tract. The present results confirm the expectation in rat that the majority of uncrossed optic axons are in fact relatively deep in the tract, though there are a number of uncrossed axons in more superticial positions. The absence of uncrossed axons at the very dorsal border of the optic tract is interesting. Driigeti commented that a few contralaterally projecting retinal ganglion cells are generated a half day earlier than the first ipsilaterally projecting cells. This may account for the pure crossed component along the dorsal edge of the rat’s optic tract, although it should be pointed out that even if the first cells generated were both ipsilaterally and contralaterally projecting ganglion cells, the difference in the length of their intraocular axonal trajectories to the optic nerve head might produce a relative delay in the arrival of the uncrossed optic axons. in the tract. The present results are consistent with the observed delay in the detection of any anterogradely transported label in the ip silateral optic tract of fetal rats following u&ocular injections of HRP.2 Axonal position in the optic tract roughly anticipates the relative position of terminal arborixations in the target visual nuclei. 9z2356 Note that the position of the uncrossed optic axons in the tract may likewise
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anticipate the arrangement of ocular lamination within the rat’s dLGN, where there are three ocular laminae, one for the uncrossed axons, surrounded by a deeper lamina and a more super&al lamina, which both receive crossed axons”
Thepresence of non-optic axons within the optic tract Tsang-‘* described the presence of non-optic, commissural, axons coursing within the optic tract of the rat, which he attributed to Gudden’s commissure. Some of Gudden’s commissure runs caudomedial to the tract, but a portion of this commissure, composed of many axons of a sixe 6ner than the optic axons, was described running through the caudal one third of the optic chiasm. ‘* Many of these fibres were traced into the ventral lateral geniculate nucleus, where in the present study retrogradely labelled neurons were observed following injections into the opposite optic tract. The present study conlirms many of Tsang’s’* acute observations, based as they were on limited histological techniques. The population of non-optic axons, though vastly outnumbering the uncrossed optic axons, is still a relative minority by comparison with the crossed optic axons. The fact that they are greatly outnumbered by optic axons, and that they are present only caudomedially, but not rostrolaterally, both indicate that their presence is not sufficient to account for the appearance of a partial segregation of the coarse from the line optic axons (preceding article*‘), nor for the appearance of a relatively deep location of the uncrossed optic axons (present results), caused by, for example, a large population of f’me-calibre axons of solely non-optic origin traversing the optic tract along its complete pial margin. Although both the HRP injections into the dorsolateral thalamus and the lesions of the optic tract and neighbouring thalamus demonstrate a population of non-optic axons within the optic tract, they show dissimilar patterns of labelling outside the tract, in the supra-optic commissures (i.e., more caudomedial labelling in the supra-optic commissures following thalamic lesions rather than injections; compare Figs 8 and 10). This is likely to be a consequence of the dissimilar populations of axons affected by the two manipulations. The HRP injections should retrogradely label those axons that terminate within the regions of the injection site and those axons transected by the syringe needle in the region of the injection site, and should anterogradely label those axons whose somas are in the region of the injection site. Axons retrogradely labelled by transection are
Fig. 10. (a) Semithin section through the optic tract opposite to the hemisphere that had received an aspirative lesion of the optic tract and dorsolateral thalamus ten days previously. Note the degenemte axonal pro6lce in the optic tract. Compare their distribution with the distribution of intact axons in Figa 2 and 5. Dorsal is up, catiomedial is to the left. 86-237L. p-Phenylene&mine stain. Bar = 50 pm. (b) Line drawing of a semithin section from the same optic tract as in (a), displaying the position of degenerate profiles following ablation of the opposite optic tract and dorsolateral thalamus.
1038
B. E. RFESE
likely to be the easiest to detect. The lesions, in contrast, should produce an anterograde degenerative response in all axons interrupted, regardless of location of either soma or terminal. The lesion is much closer to the midline than the injection sites, affecting axons that may not traverse the region of the injections. As more medial structures in the diencephalon which contribute to the supra-optic commissures send their axons through more caudomedial locations in the commissures,” then the present discrepancy between these two results would be expected. This conspicuous population of non-optic axons in the optic tract will be a major source of error for studies attempting to determine the size of the population of the crossed and uncrossed pathways from counts taken in the tract. For instance, Shirokawa and co-workers28 estimated the number of crossed and uncrossed optic axons by counting axons in the optic tract ipsilateral and wntralateral to an enucleation made 60 days earlier. Though their counts for the crossed population were only fractionally higher than other estimates of this population (based on fibre wunts in the optic nerve, or on counts of retrogradely labelled ganglion cel!s in the ntina4.‘.‘3.‘63’~“~~), their estimates for the uncrossed population were twice the number found in all other studies (based on retrogradely labelled ganglion cell~‘~~“*‘*).Furthermore, they then compared these estimates with those obtained for rats enucleated on day of birth and examined 75 days later, finding a fourfold increase in this uncrossed population, a
value seven times the estimates obtained in other studies (based on retrogradely labelled ganglion cells;‘4*‘8note that these studies obtained only a 50% increase in the number of ipsilaterally projecting retinal ganglion cells). The adult enucleation, followed by a lengthy survival period, results in significant gliosis within the tract contralateral to the removed eyem which should keep the borders of the tract well-defined. Many of the non-optic axons within the optic tract would still be mistaken for uncrossed optic axons, and would be counted in their study. An earlier removal of this crossed population, followed by a similar survival period, should prevent the normal development of spatially discrete uncrossed and non-optic populations, and prevent the formation of a well-defined caudomedial border of the optic tract, allowing many non-optic axons destined to travel both in and outside the optic tract to be mistaken for uncrossed optic axons. This may account for the tremendous elevation seen by these investigators2* in the (presumed) uncrossed population following an enucleation performed on the day of birth. Acknowledgemenm-This reseamhwassupportedbyagmnt from the Medical Research Council (PG 8324037). The author held a Junior Research Fellowhip at Womester College, Oxford, during the course of this research. It is a pleasure to thank M. Sanders for expert t&mLal assistance, T. Richards for orenarina the fiaurcs. T. Rarchav.C. Rmlscv and B. Archer ~or’pria&g thg phdmgraphs &i ehctr& micrographs, and G. Baker and R. W. Guillery for comments upon an early draft of the manuscript.
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10. lbkcr J. S., Ym P. E., Meta C. B. and RwtioniA. J. (1977) A new opedc, sensitive and noaoprcimogenic reqent for the e of bonendih proddu. Hirroehwn. 1.9.789-792. 11. He&k C. K. (1941) v of tlu optic nerver of Ambl~toma. J. camp. Neural. 74,473-534. 12. Herriek C. K. (1942) optic mul posto+c ay&mu in tha tumio of Anrblystumn rig&amr. 1. cayx N~woI. 77, 191-353. 13. Hn&A. (1977) The @gmental-mt optk nerve: film’ count and flbrc d&mater spectrum. J. camp. Nrurd 176, m the develqing redem vlaual systefn. 14. JdTeryG.(l~)Re&ulgan&mcelldeathandt&nalBeldretra&a &VI &eir, Eu. 13) U-96. 15. JdTery G., Cony A. md KuvponH. G. J. M. (1981) Rlfurating retinal gut&ion cell axons in the rat, demonstrated byrotrogrwlcdoubbLb%lay.kql)3,Rn.~3cso. 16. Lam K.. Sdbo A. J. and Rnmtt M. R. (1982) Low of axons from the epic nerve of the rat during arly postnatal development. Lkol lkalrr Res. 3.487-491.
Optic and non-optic axons in the optic tract
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17. Linden R. and Perry V. H. (1982) Ganglion cell death within the developing retina: a regulatory role for retinal dcndrita? Neumtcirnce 7, 2813-2827. 18. Linden R. and Serfaty C. A. (1985) Evidence for differential effects of terminal and dendritic competition upon developmental neuronal death in the retina. Neuroscience Is, 853-868. 19. Maggs A. and Scholes J. (1986) GliaJ domains and nerve fiber patterns in the fish retinotectal pathway. J. Neurosci. 6,424-438. 20. Mesulam M.-M. (1982) Principles of horseradish peroxidase neurohistochemistry and their applications for tracing neural pathwayz+Axonal transport, enzyme histochemistry, and light microscopic analysis. In
51, 172-178. 29. Sidman R. L. (l%l) Histogenesis of mouse retina studied with thymidine-H3. In 7’he Sfrucrure o/the Eye (cd. Smelser G. K.). Academic Press, New York. 30. Sugimoto R.. Fukuda Y. and Wakakuwa K. (1984) Quantitative analysis of a cross-sectional area of the optic nerve: a comparison between albino and pigmented rats. Expi Rrain Res. 54,266-274. 31. Torrealba F., Guillery R. W., Eysel U., Polley E. H. and Mason C. A. (1982) Studies of retinal repremntations within the cat’s optic tract. J. chem. Neural. 211, 377-396. 32. Tsang Y.-C. (1940) Supra- and post-optic commissures in the brain of the rat. J. camp. N-1. 72, 535-567. 33. Walsh C. and Guilkry R. W. (1985) Age-related fiber order in the optic tract of the ferret. J. Neurosci. 5,3061-3069. 34. Walsh C. and Polley E. H. (1985) The topography of ganglion ce-llproduction in the cat’s retina. J. Neurosci. $741-750. 35. Walsh C., Polley E. H., Hickey T. L. and Guillery R. W. (1983) Generation of cat retinal ganglion cells in relation to central pathways. Nature W2.61 l-614. 36. van Crevel H. and Verhaart W. J. C. (1963) The rate of secondary degeneration in the central nervous system. II. The optic nerve of the cat. 1. Anar., Land. 97,451-464.
(Accepted 17 February 1987)
Neurmchce Vol. 22, No. 3, pp. 1025-1039, 1987 Printed in Great Britain
THE POSITION OF THE CROSSED AND UNCROSSED OPTIC AXONS, AND THE NON-OPTIC AXONS, IN THE OPTIC TRACT OF THE RAT B. E. b@SE University of Oxford, Department
of Human Anatomy, South Parks Road, Oxford OX1 3QX, U.K.
Abstrad-The position of the crossed and uncrossed optic axons, and of the non-optic axons, within the optic tract was determined in the adult hooded rat. Horseradish peroxidase bistochemistry and lesion-induced degeneration of axonal profiles were independently used to study the position of the three relevant populations of axons witbin the optic tract. The boundaries of the optic tract are distinct at all but its caudomedial border, where it abuts the supra-optic commissures running parallel to the fibres of the optic tract. Labelling the crossed population of optic axons, or inducing their degeneration, both demonstrate a clear caudomedial border of the optic tract, although a number of optic axons stray out of the optic tract and course within the supra-optic commissures immediately caudomedial to the tract. The uncrossed optic axons are, as a population, positioned relatively deep in the optic tract, towards its dorsal border. A few occur at further ventral positions, but their density is greatly reduced there. Tbere is also a very thin region along the dorsalmost edge of the optic tract free of uncrossed optic axons. The relative position of the uncrossed to the crossed optic axons is discussed in the context of the mammalian optic tractas a chronologicalmap:spatial position in the tract may reflect temporal order of axonal arrival during early development. A large population of non-optic axons belonging to Gudden’s commissure courses within tbe boundaries of the optic tract at a relitively ventral position. They are most frequent caudomedially, and are absent rostrolatcrally. Hence, axons of the optic tract and Gudden’s commissure are substantially intermingled in the caudomedial half of tbe optic tract. These non-optic axons greatly outnumber the uncrossed optic axons, and will consequently distort counts of uncrossed optic axons based on intact pro&s that remain after removal of the opposite eye. However, they are still a minority in comparison to the crossed optic axons in this region.
There is increasing evidence to demonstrate that the mammalian optic tract can he viewed as a chronological map of axonal arrival, as has been known for various non-mammalian species for some time.6**~L1~1z1g~27 In the cat, for example, there is a correlation between the temporal pattern of genesis of the different ganglion cell types with the position of axon diameter classes across the deep-to-superficial axis of the optic tract.gJ’ls In the ferret, if the
retinofugal projections are labelled during the period of ganglion cell genesis, they are observed to occupy the whole optic tract when examined 24 h later; yet the labelled fibres occupy only deeper portions of the tract if examined 4 days later, when the tract is much larger in size due to the addition of newly arrived fibres arising from recently generated ganglion cells.” And in the fetal mouse, profdes of axonal growth cones are found in the optic tract only near its outer border, subjacent to the superficial (pial) surface.’ These various results strengthen the belief that one may read temporal order out of spatial position of the optic fibres within the mammalian optic tract.
The present investigations examined the position of the uncrossed optic axons within the optic tract of the adult rat. The ipsilaterally projecting retinal ganglion cells in the rat represent less than 5% of the total retinal ganglion cell population, comprising about 20% of the ganglion cells situated within the temporal retina. 3~15Given that this population is so small relative to the contralaterally projecting retinal ganglion cells, it should be possible to identify the position of these uncrossed optic axons relative to the complete crossed projection and, from this, to infer their relative arrival times in the optic tract during early development. The present investigation also examined the position of the non-optic axons that course amongst the optic axons within the optic tract. They comprise a non-trivial proportion of axons with respect to the population of uncrossed optic axons, and their presence is likely to account for the substantially elevated estimates of the uncrossed population when based on counts of intact axonal profiles in the optic tract following contralateral eye removal.28 EXPERIMENTAL
Abbreuiations: dLGN, dorsal lateral geniculate nucleus; HRP, horseradish peroxidaq SC, superior colliculus; TMB, tetramethylbcnzidine.
PROCEDURES
Experiments employing horseradish peroxidase hirtochemflry to reveal axonal populations Seven adult male and female Long-Evans hooded rats 1025
1026
B. E. RnsE
were used. Surgical procedures for the horseradish peroxidase (HRP) injections into the eye and along the optic tract were identical to those described previou~ly.~‘~~Briefly, each rat was anaesthetited and placed in a stereotaxic headholder. They received either a unilateral eye injection (5~1 of a 40% HRP solution dissolved in 2% dimethyl sulphoxide) or a series of six stereotaxicaliy placed intracerebral injections (0.15 ~1 of same, per injection), the coordinates of which had been previously determined to locate the optic tract. Following either a 24 or a 48 h survival, respectively, rats were heavily anaesthetized and then transcardially perfused with 5Oml of 0.9% saline followed by 300 ml of 1.25% pamformaldehyde plus 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2 at 2OT). For those rats that received mtracerebral mjections of HRP. they were additionallv net-fused with lOOm1 of 1.O% pa&fo&raldehyde in b&r *(pH 7.2 at 4°C) immediately following the saline, and their eyes were then removed, after which the perfusion was continued with the paraformaldehy~ghnaraldehyde solution. Brains were removed and left to sink overnight in 2O% sucrose dissolved in buffer (4°C). Retinal fiatmounts were prepared and reacted for HRP histochemistry, as bef0re.u Brains were cut either coronally or horizontally at 5Opm on a freezing microtome. Sections were reacted for enzymatic activity using tetrametbylbcnxidine (TMB) or both TMB and pphenyknediamine plus catechol (alternate sections) as the chromogen(s) toXs Sections through the optic tract were. drawn with the aid of a drawing tube attached to the microscope at a final magnification of x 50. Experimen:s profles
to show the distribution o/&generaring
axonal
Six adult mak and femak Long-Evans hooded rats were used.Eachmtwasanaen&etizsdandpkcedinastereotaxic headholder. They received either a tmikteral emtckation or a surgical ablation of one optic tract. For the enuckations, the globe was removed in its entirety, the orbit Packed with gelfoam, and the eyelids sutured. For the optic tract transections, the Jcplp was opened, the doraolateral skull was removed, and an aspirative lesion was made under v&al control with the aid of an operating microscope. The overlying cortex and hippoeampLIs were at&ted to reveal the donolateral aspect of the optic tract. This was then aspiratedtoremovetheentirelateralgcnkukten~andto sever the entirety of the optic tract. The cavity was then
filkdwithgelfoamandthesealpsutured.Allratswerekft to survive for 9 or 10 days. They were then perfused. and their optic nerves and tracts were pmcemed for hgbt and ekctron microscopy. using procedures identical to those described in the pmceding pa~er,~ except that (I) only the intmcranialopticneNeawereprouamd,and(2)tbehamispbem containing the ablated optic tract was pkeed into 30% sucrose in 10% formalin, cut coronnBy at 5Opm on a freezing microtome, and a l-in-5 series was mounted and stained with Cmsyl Viokt acetate. Se&thin sections of the optic tract ipailateml to an enucknted aye or opposite to an abkteddomokteraltbakmuswemdrawnat x4tlOwiththe aidofadrawingtubeattachedtothemicroscope.andthe position of wte profike was inttiatted. All figurea and photomkrogmpha of uprimDnd dcgenarationandofintactaltmlttlpfol!&ailbWatamamtial inapkneorthogonaltothedimdiondthean0ea,and conaequantly do not cortwpond to any amndard berso4utc plane.Themmabdng6gttmsandpho~~~ 1, 7,8and9)showsectionscutinthecoronaipkne. In the optic tract contmlateral to the ettuckation of one rat, axon dknmtem were measured ffom &CtrCm micrographs for evuy intact axon contaisbad within a @on 543gm’, and from tbeae WtaaxonW distributions were conatmcted for a mmtber of loatb~ across the optic tract and supra-@c commksuru. These
procedures were identical to those described in the preceding paper.2’ RESULTS
The crossed and uncrossed
optic
axons
in the optic
tract
The intraocular injections of HIW produced homogeneous filling of the target visual nuclei, in particular the dorsal lateral geaiculate nucleus (dLGN) and superior colliculus (SC), with no evidence for a failure to label a portion of the retina hecause of insmiicient spread of the tracer in the eye or because of damage to the axonal bundles in the optic fibre layer. Figure 1 shows photomicrographs and line drawings of coronal saxions through the optic tract contralateral and ipsilateral to an eye injected with HRP. Contralaterally. the labcUing is darw and even throughout the optic tract (Fig. lb and d). Along the medial edge of the tract there is a region of white matter devoid of all but a few lab&d axons and, medial to this unlabelled region+ there is a separate population of labelled axons. The latter, labelled, axons form the accessory optic tract, coursing along the ventral surface of the brain to the medial terminal nucleus of the accessory optic system. The unlabelled axons between the accessory and main optic tracts contain the various supra-optic commiaaures and other non-commissural fibre pthways.n In the ipsilateral optic tract, labelled optic axons are found relatively deep in the optic tract, nearer its dorsal border (Fig. la and c). The labelled axons occupy this deep position throughout the length of the tract, beginning dorsally just behind the optic chiasm, and continuing in a dorsomedial position as the optic tract courses doraolaterally over the diencephalon. In coronal sections, there is a small region free of uncrossed axons at the very dorsal and lateral extremes of the tract, along the tract’s deep border (Fig. la and c). The distribution of the crossed and uncrossed optic axons was largely confirmed by the location of degenerate profiles observed in the optic tracts ipsilateral and contralateral to an enu&ation. Figure 2 is a photomicrograph of the optic tract ContU to the enucleation. Degenerate pro&s are okserved caudomedially in the accessory optic tract (arrowheads); rostrolateral to this, intact axonal pr06ks Further roatroform the supra-optic corm&axes. laterally, the optic tract is dktinct at all but its caudomedial boundaries, where Gwkbed’s cammissure runs adjacent to the. optic axons. A few optic axons stray out of the region of &rMy pIcked degenerate pro&s, but a rough border can still be discerned (broken tine in Fig. 2). In the tract ipsilateral to the enuclation (Fig. 3), degenerate pro&s are found ptimarily deep in the tract, removed from the pial surface. Ssms i&Meral axons can be found at relatively soperiichl krcations
Fig. 1. Photomicrographs [(a} and fb)] and line drawings [(c) and (d)] of coronal sections through the optic tract ipsiiateral [(a) and (c)] and contralateral [@) and (d)] to a unilateral intra-oculax injection of HRP. Spacing between the sxxtions in (c) and (d) is 400 pm, with rostral sections to the top. Dorsal is up, medial is to the centre. (a), (b) 86-38. Bar = 100 pm. (c), (d) 86-37.
b
dorsal
caudo-medial
J
Fig. 2. 1028
opticand non-opuc axons in the optic tract (Fig. 3b), but the vast majority are nearer to the tract’s dorsal border. The degenerate profiles secll dorsally tend to be conspicuously larger than those observed ventrally, as might be expected from the axon diameter sixes described in these two regions of the tract in the preceding article.u very few degenerate pro&s are found immediately adjacent to the tract’s dorsal border (Fig. 3a and b). It was expected that this pattern of degeneration in the tract ipsilateral to enucleation (Fig. 3) would be detected by the complementary pattern of intact axons seen in the tract contralateral to enucleation (Fig. 2). Indeed, many intact large and small diameter axons were observed in this very dorsal region of the contralateral optic tract (Fig. 2a). But in addition there were a surprising number of intact axonal profiles, most of a smaller diameter, found primarily caudomedially in the tract (Fig. 2b). Figure 4 displays electron micrographs from three regions of an optic tract contralateral to an enucleation. Dorsally, deep in the optic tract, both some large and small diameter axons, though relatively few in number, appear intact (Fig. 4a), while caudomedially many more intact profiles ate present, though of smaller diameter (Fig. 4b). Rostrolaterally, very few unambiguously intact profiles are observed (Fig. 4~). Figure 5 displays the distribution of the axon diameters of these intact profiles at various locations across the optic tract contralateral to an enucleation, for every axon contained within regions 543 pm*. Large and small axons are found in that region of the tract known to contain degenerating profiles when the ipsilateral eye is removed (Fig. 5, histograms l-4; see Fig. 3), the coarser axons comprising a much greater proportion of the total than seen in the population of crossed, or crossed plus uncrossed, optic axons @receding pape?‘). Yet the number of intact axons here is insignificant in comparison with the large number of intact axons of small diameter (2 pm or less) in the caudomedial region of the optic tract (histograms 10-14, 17 and 18). Here, the definition of an intact axonal profile is based on the absence of those characteristics typical of degenerating axons: darkening of the axoplasm, disintegration of axoplasmic organdies, empty regions within the axon, and/or a disruption or shredding of the myelin lamellae. These features are rarely present in material examined from normal, nonexperimental, rats. The presence of these features is dependent upon survival time and on the diameter of the axon in question.M Hence, the failure to detect
1029
such protbes may not be a reliable indication that a given axon did not arise from the enucieated eye. However, this seems an unlikely explanation for the preset~e of these intact axons, since the axon diameter distribution in this region of the tract (caudomedUy) does not dilfer from locations rostrolaterally (see preceding article), where virtually all axonal profiles are degenerate following enucleation (Fig. 2c, 4c, Fig. 5, histograms 6-9, 15 and 16). Furthermore, few unambiguously intact axons can be found in the optic nerve of the enucleated eye ten days following enucleation (Fig. 6). For these reasons, it seems likely that these intact axons in the tract do not come from the contralateral retina. These same reasons make it unlikely that the intact axonal pro&s seen caudomedially arise from the ipsilateral retina but fail to transport significant amounts of HRP and do not show a degenerative response 10 days following enucleation. Nevertheless, the following experiments were undertaken to demonstrate that there is a population of commissural, non-optic, axons in the same region of the optic tract that contains these intact axonal profiles subsequent to removal of the contralateral eye. The non-optic axons in the optic tract Figure 7 shows a typical injection of HRP into the optic tract and dorsolateral thalamus. The injections of HRP along one optic tract produced heavy labelhng of the contralateral and ipsilateral retinal ganglion cell populations. The complete contralateral retina was labelled, as was the ipsilateral retina’s temporal crescent, and many of the cells displayed a Golgi-like labelhng indicative of uptake of the tracer by axons that were damaged by the syringe needle. These injections of HRP consistently labelled axonal processes in the contralateral optic tract. Figure 8a is a photomicrograph displaying label in the opposite optic tract typical of the present series of rats. In coronal sections, the labeiled axons occupy a ventral, pial position, and can be followed caudally in a progressively dorsomedial position near the tract’s caudomedial border (Fig. 8b). Although this population of labelled axons of the supra-optic conunissures cannot be readily defined with respect to the caudomedial border of the optic tract in this material, by comparing these results (Fig. 8) with those showing the crossed optic axons (Fig. l), it is clear that a large majority of these labelled profiles are well within the limits of the “optic” tract. This regional localization of non-optic axons
Fig. 2. Semithin section through the optic tract contralateral to an cnuckation ten days previously. Note the intact axonal profiles doraally (a), and aho~au~Iomedially (b), within the optic tract. Also note the &generate prohles at the far kft of the photormaogroph, in the accessory optic tract (arrowbatds). The broken line indicati the caudomedialborderof the optic tmct. (a)-&) Detaila from the re@onrindicated in the main photogrpph,dorsally. caudo~y and mstrokterally, mspcetively,from a nearbytion. 86341. p-I%enylcncdiaminestain. Bar = 50 pm for the main photorpaphand 25 pm for (a)-(c).
T
Fig. 4. Electron micrographs from the optic tract contralateral to an enucleation ten days previously. (a)+) Dorsal, caudomedial and rostrolateral optic tract, respectively, as in Fig. 2. 86-341. x6100. 1031
1032
B.E. REF~
O
E~ _= ..=
8~
~8.o E~
O
O
Optic and non-optic axons in the optic tract
Fig. 4. Electron micrograpb of an optic nerve ipsiiatersf to an enucleationten days previously.Not6 that virtually all of the axonal proNes appear degenerate. 86412L. x 6100.
within the optic tract was confirmed by the present of degenerate profiles following ablation of the opposite optic tract. Figure 9 displays a coronal section typical of the damage produced in two rats receiving ablations of the optic tract. At this coronal level, rostrai to the lateral genicuiate nuclei, the extent of damage to the dorsolateral thalamus can be seen, which extends caudally to the medial geniculate
nucIeus. The optic tract in this section (arrowhead in Fig. 9) is heavily gliotic, being completely transected further caudally. Figure 10 shows the pattern of degenerate axonal profiles following ablation of the opposite optic tract. The degenerate axons are positioned ventrally and caudomedially, both within the optic tract, and in the supra-optic commissures. Degenerate profiles are ab-
Fig. 7. Photomicrograph of a coronalsa%iondis&ying n typical injection a&eof heish pemxidase into the optic tractand dorsokitcral thalamus in the pnsent series of rats. 84-552.CresyI Violet acetate counterstain. Bar = I mm.
1033
b
Ontic and non-optic axons in the optic tract
1035
Fig, 9. Photomicrograph of a coronal section through the hemisphere that had been given an aspirative lesion of the dorsolateral thalamus. The arrowhead indicates the optic tract, in this section rostra1 to its transection. 86-237. Cresyl Violet acetate stain. Bar = 1mm.
sent from the deep, dorsal, part of the optic tract, where the majority of uncrossed axons course, and
from the rostrolateral optic tract, where no intact axonal profiles are observed following contralateral eye removal. Although there are many degenerate profiles in the caudomedial optic tract, Figs 2 and 10 indicate that the vast majority of axons in this region arise from the contralateral retina. Degenerate profiles in Figs 3 and 10 together, then, account for the distribution of intact axons seen in the optic tract after enucleation of the contralateral eye (Fig. 2). DISCUSSION
The present study has
The position of the uncrossed optic axons In the cat and monkey, the distributions of the crossed and uncrossed optic axons in the optic tract are almost completely overlapping, nearly filling the full extent of the optic tract’s boundaries. The exception in the cat concerns a small pure crossed portion of the tract, situated dorsolaterally, in that part of the optic tract representing inferior retina.” This discrepancy between the domains of the crossed and uncrossed optic axons has been interpreted in terms of the dissimilar periods of ganglion cell genesis
b
c Fig. 10. 1036
Optic and non-optic axons in the optic tract
for cells in the inferior nasal (contralaterally projecting) and inferior temporal (ipsilaterally projecting) retina. The absence of this pure crossed region ventromedially (the other extreme along the deep border of the cat’s optic tract), where the tract represents superior retina, corresponds well with the less discrepant periods of retinal ganglion cell genesis in superior nasal and superior temporal retina.9 In the rat, the present results describing the position of the uncrossed optic axons, arising from a proportionately smaller number of ipsilaterally projecting retinal ganglion cells, may be interpreted in the context of the optic tract as a chronological map (see introductory paragraphs). Although the relevant birthdating studies have not been done in the rat, studies of ganglion cell genesis in the mouse may have sutlicient generality for the temporal order of events in ratsSJg Ipsilaterally and contralaterally projecting retinal ganglion cells in the mouse are generated between days E-l 1 and E-17, but the contralaterally projecting cells continue to be generated until E-19.’ The peak production period for the ipsilaterally projecting cells is around E-13114.’ One might expect then that the bulk of the uncrossed optic axons would tend to occupy a relatively deep position in the optic tract, and that there would be few, if any, uncrossed optic axons along the pial surface of the tract. The present results confirm the expectation in rat that the majority of uncrossed optic axons are in fact relatively deep in the tract, though there are a number of uncrossed axons in more superticial positions. The absence of uncrossed axons at the very dorsal border of the optic tract is interesting. Driigeti commented that a few contralaterally projecting retinal ganglion cells are generated a half day earlier than the first ipsilaterally projecting cells. This may account for the pure crossed component along the dorsal edge of the rat’s optic tract, although it should be pointed out that even if the first cells generated were both ipsilaterally and contralaterally projecting ganglion cells, the difference in the length of their intraocular axonal trajectories to the optic nerve head might produce a relative delay in the arrival of the uncrossed optic axons. in the tract. The present results are consistent with the observed delay in the detection of any anterogradely transported label in the ip silateral optic tract of fetal rats following u&ocular injections of HRP.2 Axonal position in the optic tract roughly anticipates the relative position of terminal arborixations in the target visual nuclei. 9z2356 Note that the position of the uncrossed optic axons in the tract may likewise
1037
anticipate the arrangement of ocular lamination within the rat’s dLGN, where there are three ocular laminae, one for the uncrossed axons, surrounded by a deeper lamina and a more super&al lamina, which both receive crossed axons”
Thepresence of non-optic axons within the optic tract Tsang-‘* described the presence of non-optic, commissural, axons coursing within the optic tract of the rat, which he attributed to Gudden’s commissure. Some of Gudden’s commissure runs caudomedial to the tract, but a portion of this commissure, composed of many axons of a sixe 6ner than the optic axons, was described running through the caudal one third of the optic chiasm. ‘* Many of these fibres were traced into the ventral lateral geniculate nucleus, where in the present study retrogradely labelled neurons were observed following injections into the opposite optic tract. The present study conlirms many of Tsang’s’* acute observations, based as they were on limited histological techniques. The population of non-optic axons, though vastly outnumbering the uncrossed optic axons, is still a relative minority by comparison with the crossed optic axons. The fact that they are greatly outnumbered by optic axons, and that they are present only caudomedially, but not rostrolaterally, both indicate that their presence is not sufficient to account for the appearance of a partial segregation of the coarse from the line optic axons (preceding article*‘), nor for the appearance of a relatively deep location of the uncrossed optic axons (present results), caused by, for example, a large population of f’me-calibre axons of solely non-optic origin traversing the optic tract along its complete pial margin. Although both the HRP injections into the dorsolateral thalamus and the lesions of the optic tract and neighbouring thalamus demonstrate a population of non-optic axons within the optic tract, they show dissimilar patterns of labelling outside the tract, in the supra-optic commissures (i.e., more caudomedial labelling in the supra-optic commissures following thalamic lesions rather than injections; compare Figs 8 and 10). This is likely to be a consequence of the dissimilar populations of axons affected by the two manipulations. The HRP injections should retrogradely label those axons that terminate within the regions of the injection site and those axons transected by the syringe needle in the region of the injection site, and should anterogradely label those axons whose somas are in the region of the injection site. Axons retrogradely labelled by transection are
Fig. 10. (a) Semithin section through the optic tract opposite to the hemisphere that had received an aspirative lesion of the optic tract and dorsolateral thalamus ten days previously. Note the degenemte axonal pro6lce in the optic tract. Compare their distribution with the distribution of intact axons in Figa 2 and 5. Dorsal is up, catiomedial is to the left. 86-237L. p-Phenylene&mine stain. Bar = 50 pm. (b) Line drawing of a semithin section from the same optic tract as in (a), displaying the position of degenerate profiles following ablation of the opposite optic tract and dorsolateral thalamus.
1038
B. E. RFESE
likely to be the easiest to detect. The lesions, in contrast, should produce an anterograde degenerative response in all axons interrupted, regardless of location of either soma or terminal. The lesion is much closer to the midline than the injection sites, affecting axons that may not traverse the region of the injections. As more medial structures in the diencephalon which contribute to the supra-optic commissures send their axons through more caudomedial locations in the commissures,” then the present discrepancy between these two results would be expected. This conspicuous population of non-optic axons in the optic tract will be a major source of error for studies attempting to determine the size of the population of the crossed and uncrossed pathways from counts taken in the tract. For instance, Shirokawa and co-workers28 estimated the number of crossed and uncrossed optic axons by counting axons in the optic tract ipsilateral and wntralateral to an enucleation made 60 days earlier. Though their counts for the crossed population were only fractionally higher than other estimates of this population (based on fibre wunts in the optic nerve, or on counts of retrogradely labelled ganglion cel!s in the ntina4.‘.‘3.‘63’~“~~), their estimates for the uncrossed population were twice the number found in all other studies (based on retrogradely labelled ganglion cell~‘~~“*‘*).Furthermore, they then compared these estimates with those obtained for rats enucleated on day of birth and examined 75 days later, finding a fourfold increase in this uncrossed population, a
value seven times the estimates obtained in other studies (based on retrogradely labelled ganglion cells;‘4*‘8note that these studies obtained only a 50% increase in the number of ipsilaterally projecting retinal ganglion cells). The adult enucleation, followed by a lengthy survival period, results in significant gliosis within the tract contralateral to the removed eyem which should keep the borders of the tract well-defined. Many of the non-optic axons within the optic tract would still be mistaken for uncrossed optic axons, and would be counted in their study. An earlier removal of this crossed population, followed by a similar survival period, should prevent the normal development of spatially discrete uncrossed and non-optic populations, and prevent the formation of a well-defined caudomedial border of the optic tract, allowing many non-optic axons destined to travel both in and outside the optic tract to be mistaken for uncrossed optic axons. This may account for the tremendous elevation seen by these investigators2* in the (presumed) uncrossed population following an enucleation performed on the day of birth. Acknowledgemenm-This reseamhwassupportedbyagmnt from the Medical Research Council (PG 8324037). The author held a Junior Research Fellowhip at Womester College, Oxford, during the course of this research. It is a pleasure to thank M. Sanders for expert t&mLal assistance, T. Richards for orenarina the fiaurcs. T. Rarchav.C. Rmlscv and B. Archer ~or’pria&g thg phdmgraphs &i ehctr& micrographs, and G. Baker and R. W. Guillery for comments upon an early draft of the manuscript.
1. Boloventa R. aad Mason C. (la pnss) Growth cone morphology varies with P&ion in the &v&ping mouse visual pathway Ram retina to 5st targets. 1. New-ok. 2. Bunt S. M., Lund R D. and Land P. W. (1983) Pramtal dcvdopment of the optic projection in albino and boo&d nts. Lkvl Brain Rrs. 6,149-168.
3. Cowey A. and Perry V. H. (1979) The lxoja&n of the temporal ntina in rats, studied by retrograde tmrqort of homera&& lreroxidase. EdpI WIpdnRes. 3s, 457-464. 4. Crapo D., O’Leary D. D. M. and Cowan W. M. (1985) Changse in the numbers of optic nerve &era duriag late peaatal aml $umaaml dew+ment in the alllino lat. Ball Brain Res. 19,129-134. 5. DriOrU.C.(l~~)Birta~of~~alhBiviogriretotbecroad~~ qXicl%ojuzionsin the mouse. Proc. R. sot. m 57-n. 6. ButcrS.S.,RproirA.C.mdltirhP.E.(198l)Thegmwthand~tionoftheopticnaveandcractinjuvmik and adult griidtU. J. Ncy~os~i.1, 793-U 1. Formeter J. and R&s A. (1967) Nave filma in the optic QCXVC of rat. NoMe. Lmd U4,245-247. tadneles. ;I: Chz~ R M. aad Orrrnt P. (1978) The diem~&& course of mlmnemtinR mtinoteetal mbers in xw J. Embryoi, exp. Morph. 4&‘201-216. 9. Guiky R. W.. Polley E. H. and Torrulba F. (1982) The srrcnqcmcnt of axona accotxiin~to fiber dii in the optic tmct oftbe at. J. Newmci. 5714-721.
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1039
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(Accepted 17 February 1987)