experimentally induced enlargement of the uncrossed retinotectal pathway in rats

7/28/2019 Experimentally Induced Enlargement of the Uncrossed Retinotectal Pathway in Rats

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Brain Research, 211 (1981) 37-570 Elsevier/North-Holland Biomedical Press 37

EXPERIMENTALLY INDUCED ENLARGEMENT OF THE UNCROSSEDRETINOTECTAL PATHWAY IN RATS

L. S. JEN and R. D. LUND*University ofHong Kong, Hong Kong (Hong Kong) and (R.D.L.) Department of Anatomy, MedicalUniversity of South Carolina, Charleston, S.C. 29403 (U.S.A.)(Accepted May 22nd, 1980)Key words: plasticity - uncrossed optic pathway - retinotectal pathway

SUMMARY

The present experiments investigate the effects of neonatal lesions upon projec-tion patterns of the uncrossed retinotectal pathway in albino rats. The results indicatethat enlargement of the terminal field from one eye can be induced either by a contra-lateral optic tract lesion or by removal of the opposite eye at birth. The extent of theenlargement is more prominent in the latter case. If an optic tract lesion isaccompanied by eye enucleation on the side ipsilateral to the tract lesion, theuncrossed retinotectal projection from the remaining eye will undergo furtherenlargement. However, optic fiber counts show that such an enlargement of theterminal field is not due to a significant increase in the number of uncrossed opticaxons which contribute to the enlarged projection, but rather to an increased terminalarbor of individual axons (as shown by results from fiber counts). While a severeganglion cell loss was observed in the retina contralateral to a tract lesion, asubstantial population of cells persists in the ganglion cell layer and the number ofcells appears much higher than the number of uncrossed optic axons arising from thesame eye. The implications of these findings are discussed in relation to resultsreported in previous studies.

INTRODUCTIONIt has been shown in the hamster 40341hat a unilateral tectal lesion made at birth

results in the formation of an aberrant recrossed retinotectal pathway and sprouting ofoptic axons in the nucleus lateralis posterior (LP). The extent of the recrossed pathway

* To whom reprint requests should be addressed.


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38bears a reciprocal relation to the amount of sprouting in LP. Based on theseobservations, Schneider et al.12,40,41 proposed that the formation of anomalous axonalconnections reflects the tendency of growing neurons to conserve their total quantityof axonal arborization. The present study is intended to test this hypothesis further bystudying another feature of the retinotectal pathway. It is known in normal albino ratsthat the retinotectal pathway is predominantly crossed and that the uncrossedprojection is very small and restricted mainly to the anteromedial portion of thestratum opticum of the ipsilateral superior colliculus27Ji. However, if one eye isremoved early in development, the uncrossed retinotectal pathway from the remainingeye will enlarge its terminal field to occupy the entire area of the ipsilateral colliculusss?so. Since Cunningham et al.s*gJO have shown that at least some of the ipsilaterallydirected axons are collateral branches of axons which also project contralaterally, wewondered whether cutting the crossed projection by a tract lesion would lead tocompensatory enlargement of the terminal field of the uncrossed axons, as might beexpected according to the hypothesis proposed by Schneideras. Recent work by Landand Lund24 has added a further aspect to this experiment by showing that there isalready at birth a substantial uncrossed retinotectal pathway which normally retractsin the first postnatal week, unless the opposite eye is removed, in which case it persists.These observations imply that crossed optic axons from an eye can exert an inhibitoryeffect on the growth of uncrossed axons from the opposite eye. The question thenarises as to how this effect may relate to altered growth patterns which result fromtract lesions. These matters have been investigated in the present series of experimentsin which one eye and one optic tract are destroyed in the same or separate animals.The experimental details are summarized in Table I and Fig. I.MATERIALS AND METHODS

The study was undertaken on Sprague-Dawley albino rats obtained fromTylerslaboratory in Bellevue, Wash. As shown in Table I, 4 experimental groups were used.The first lesion was made on the day of birth, and the second lesion was performed atthe age of 1-2 months postnatal. Further details of the experimental procedure aredescribed below.Experimental lesions

All lesions including those on neonatal animals were performed under etheranesthesia. Eye enucleation was made by separating the eyelid and gently pulling theeyeball outward so that it could be removed from the orbit by cutting the surroundingconnective tissue and extraocular muscles in addition to the optic nerve head. Thepurpose of the thalamic lesion as performed in experiments C and D (Table I) was tocut the optic tract on one side, so depriving the superior colliculus of normal retinalinput. Since retinal axons to the superior colliculus do not travel in a discrete bundlelgand since there was apparently some plasticity in optic fiber growth after blockingtheir natural course in development, it was found necessary to make an extensivethalamectomy in order to destroy this pathway. Even after a large lesion such as that


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39

A f3Fig. 1. Schematic diagram illustrating development of the uncrossed retinotectal pathway in normalanimal (A) and animals which received various lesions (B, C and D) made at birth. Unilateral thala-mectomy and eye enucleation were performed in B and C respectively, whereas both lesions were madein D. Structures removed are indicated by dotted lines. The first diagram at top illustrates retinotectalprojection pattern at birth. Note a substantial uncrossed retinotectal pathway can be observed at thisage. The uncrossed pathway retracts during normal development but persists or enlarges under variousexperimental conditions (B, C and D) despite a difference in the number of uncrossed optic axons in-volved (second and bottom rows, also see Table II). The number of uncrossed optic axons in normalanimal (A) has not been determined and is therefore marked by a question mark. In A and C (bottomrow), only the crossed branches of the optic axons are illustrated in the fiber tracts (stippled area).*Indicates right eye which gives rise to the uncrossed pathway studied in all experiments. The plus andminus signs indicate hypothetical growth promoting or inhibiting substances secreted by nerve terminalsfrom axons of different origins.

shown in Fig. 2, some axons still reached the tectum. No direct pathway could bedetected, however, in half of the animals in experiments C and D. Because of theseverity of the initial thalamic lesion, mortality was high and could be as much as100 % in some litters. Nevertheless, a total of 20 animals with complete or substantialloss of the retinal projection to the lesioned side was obtained in experiments C and D.In all cases, the thalamic lesion was made on the left side of brain.


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40TABLE IOutl ine of experimental rocedure of experiment IIIExperiments First lesion (day 0)ABcD

None Right eye enucleation 5Left eye enucleation Right eye enucleation 5Left thalamectomy Right eye enucleation IOLeft thalamectomy and

left eye enucleation Right eye enucleation IO

Second lesion (days 30-60) No. ofanimalsused

Projection patterns of the right ipsiluteral retinotectal pathwayAfter a survival time of one to two months, the right eye from some animals in all

4 experimental groups was enucleated 3-4 days before the rats were sacrificed to testthe projection of that eye using degeneration methods. The animals were perfused withbuffered 4 /o paraformaldehyde and the brains removed and post-fixed in the fixativefor several days before being transferred to 30 7: sucrose-fixative. Transverse sectionscut at 26 pm were stained according to the Fink-Heimer techniqueIs. The centralprojections of the right eye were studied with special attention being paid to theipsilateral retinotectal pathway.Fiber counts in the optic nerves and tracts

Optic nerves and tracts from rats as !isted in Table II were removed afterperfusion with 4 ,/, paraformaldehyde and postfixation with 2 :, buffered glutaralde-hyde for one hour. They were then washed with phosphate buffer several times beforebeing osmicated with 2.5 1: osmium tetroxide in buffer. The tissues were dehydratedand embedded with Epon 812 or DER. Thick and thin sections were collected andexamined with light microscope and JEOL 1OOB electron microscope, respectively.Myelinated fibers were counted in the right optic nerves and tracts in two animals fromexperiments C and D, and the right optic tract from an animal in experiment B on aseries of electron micrographs which cover whole nerves or tracts at a primarymagnification of 2000-3000 times. Fig. 3 is an example showing the optic nerve fromanimal 79B (Table II) which had received a complete tract lesion. The number ofunmyelinated fibers in the right optic nerve of one animal from each group wasestimated by counting the proportion of myelinated and unmyelinated axons (Fig. 4)in systematically selected fields observed at a magnification of 8000 times or higher.Axons without a myelin sheath, but showing a sub-axolemmal electron density andsurrounded by glial cytoplasm, are considered sectioned at a node of Ranvier and arenot included in the unmyelinated fiber count,*.Ganglion cell counts and measurements

The right eye from some rats in each experiment listed in Table I was removed 3or 4 days before sacrifice. The left eye from those animals which received a thalamiclesion alone was also removed immediately before perfusion. The temporal pole ofeach eye was marked by a suture at the corneoscleral junction for orientation after


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A

Fig. 2. Photomicrograph showing a large lesion which removed most of the thalamus and cerebralpeduncle (A) but failed to obliterate the crossed retinal projection from the opposite eye. Degeneratingaxons in a small fiber bundle in the remnant tissue marked with a square is shown in B. Fink-Heimerstain. A x 17, B x 225.


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Fig. 3. Thick Epon section showing uncrossed optic axons in an optic nerve contralateral to a thalamiclesion which obliterated completely the crossed optic projection from the same eye (animal 79F, seeFig. 1B and Table II). x 440.Fig. 4. Electron micrograph showing a small region of an optic nerve from a normal animal. Note thepresence of unmyelinated axons (arrows) among the myelinated optic fibers. x 15,000.


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,j ,. 43being removed from the orbit. After fixation in 4 % paraformaldehyde for about anhour, the cornea, lens and vitreous humor were removed and the eye cup was left in thefixative for another 30 min. It was then transferred to 5 % sucrose-phosphate bufferfor several hours before the retinas were dissected out and flat-mounted onto gelatin-coated slides. The retinas were then air-dried and stained with cresyl violet. The exactpattern and number of cells in the retinal ganglion cell layer in 12 fields, 121 ,um indiameter in the lower temporal quadrant of the retina, were reproduced by means of adrawing tube using 100 x oil immersion. The lower temporal quadrant of the retinawas chosen for studying the ganglion cell pattern because it has been shown that thenormal ipsilateral retinotectal pathway arises mainly from this quadrant of the retinass.This region is also a major source of cells contributing to the enlarged ipsilateralretinotectal pathway after lesions and is thus of particular interest in studying thebehavior of the uncrossed pathway under various experimental conditions. All cells inthe ganglion cell layer, except those small and darkly stained cells with less distinctdifferentiation between nucleus and cytoplasm, are considered as neurons20J7,45. Thesize of the cells was measured by ring-matching method, matching the boundary oftheir somata with rings of 7 different sizes. The smallest cells have a somal diameterequal to or less than 6 pm and are designated category 1, whereas the largest group hassomal diameter equal to or larger than 23 pm and is classified as category 7 (see Fig.7).RESULTS

The results are described separately for each experimental approach.Projection patterns of the uncrossed retinotectal pathway from the right eye

Results from degeneration experiments confirm previous studies that in thealbino rat the normal uncrossed retinotectal pathway is a small pathway terminatingmainly in the anteromedial part of the stratum opticum of the superior colliculus(Figs. lA, 9A and 9C). After removing one eye at birth (Fig. lC), the uncrossedpathway from the remaining eye expands its terminal field, projecting over the entirearea of the ipsilateral colliculus and invading the superficial laminae of the colliculusdeafferented by the eye enucleation. This is illustrated in Fig. 10A and 1OCwhich showthe projection pattern revealed by Fink-Heiner method. As described by Lund et a12s,the expanded uncrossed pathway is heaviest in the anteromedial portion of thesuperior colliculus but decreases substantially towards the lateral and caudal parts ofthe colliculus.

In animals with a unilateral thalamic lesion destroying the left optic tract, butwith both eyes intact (Fig. IB), the uncrossed retinotectal pathway from the right eye(contralateral to the lesion) differs from normal animals by enlarging to cover most ofthe area of the ipsilateral colliculus. The difference between this case and eye enucleatedrats is that axons of the enlarged uncrossed pathway are restricted primarily to thestratum opticum with only occasional degenerating fibers in the deeper part of thestratum griseum superficiale. Fig. 9B and 9D show examples of degeneration in the


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Fig. 5. Photomicrographs showing ganglion cell pattern in comparable regions of the lower temporalquadrant of the right retina from a normal animal (A) and an animal which received a left thalamiclesion at birth (B). Note there is overall reduction in number and size of cells in B as compared with A.Cresyl violet stain. x 450.


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45

LFig. 6. Diagram showing the 12 areas in the lower temporal quadrant of the retina where ganglion cellcounts and measurements were done. L. N, T and U indicate lower, nasal, temporal and upper pole ofthe retina. OD indicates optic disc.anteromedial region of the colliculus where density and extent of the enlargedprojection is invariably the heaviest.

If a left tract lesion is accompanied by left eye enucleation (Fig. lD), the terminalfield of the uncrossed retinotectal pathway from the remaining right eye is greatlyenlarged and appears much heavier than that observed in either of the other situations.This is demonstrated in Fig. 10B and IOD which illustrate the projection pattern in theanteromedial portion of the colliculus where the density of degeneration is heaviest. Itis necessary to point out, however, that a thalamic lesion failing to obliteratecompletely the left optic tract results in a reduction in the size and extent of theuncrossed pathway from the right eye. Such reduction has been observed in animalswhich received incomplete tract lesion, with or without eye enucleation, and isillustrated in Fig. Il.

To summarize, the results from degeneration experiments indicate that the sizeand extent of the uncrossed retinotectal pathway from one eye increases progressivelyafter obliteration of that eyes crossed projection, after removal of the opposite eye, andis most prominent after both procedures. The size of the uncrossed pathway variesdepending on how complete the tract lesion is in eliminating the crossed opticprojection.Fiber counts in the optic nerves and tracts

In order to determine whether enlargement of the terminal field of the uncrossedretinotectal pathway as shown by degeneration methods is accompanied by anincrease in the number of the uncrossed axons, myelinated fibers in the uncrossed opticpathway were counted in some of the animals from each of the different experiments.As shown in Table II, the number of myelinated fibers was determined only in opticnerves and tracts which contain strictly uncrossed optic axons. The number ofuncrossed axons in the optic nerve in normal and unilaterally enucleated animals andin the optic tract of normal rats and animals which received a tract lesion alone wasnot counted due to difficulty in distinguishing the uncrossed axons from the crossedones in these fiber tracts. The total number of myelinated fibers in the optic nerve of


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46


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47normal albino rats has already been reported (117,00014 and 130,000~1) and so suchcounts were not repeated in this study. The proportion of unmyelinated fibers presentin the optic nerve was calculated in one animal from each experimental group, andwhere possible the total number of unmyelinated axons in the optic nerve wasestimated. The results are summarized in Table II.

Unmyelinated axons are present in pigmented as well as albino animals, but donot appear to degenerate even 3 months after eye enucleation, raising the possibilitythat they are not in fact of retinal origin. Of previous studies investigating the fibercomposition of the rats optic nerve, only that of Sima and Sourander2 hascommented on the presence of unmyelinated axons, and even these authors gave noestimate as to the proportion of such fibers.

Data presented in Table II indicate that there is a substantial population ofunmyelinated axons in the optic nerve of both normal and experimental animals. Theproportion of unmyelinated axons present in the optic nerve of normal animals and

580loo (335.7) 392(346.72)

I234667 11234567 1234667LTL L;(R)Fig. 7. Comparison ofcell counts and measurements in lower temporal quadrant of retinas from animalsin different experimental situations. The sizes (somal diameter) of the 7 categories of cells (l-7) are6, 8.5, 11, 13.5, 16.5, 20 and 23 pm. Histogram A represents data obtained from right retinas fromnormal animals. Data obtained from the remaining right (R) retinas in animals which received a lefttract lesion together with enucleation of the left eye (LT + LE) during the first postnatal day is shownin histogram B. Histograms C and D are results taken from left (L) and right (R) retinas from animalswhich received a left tract lesion (LT) at birth. Each of the 7 categories of cells in histograms B, C and Dis compared with corresponding category in histogram A. Values shown in the histograms representthe mean of somal diameter f S.D. The abscissa and ordinate signify the sizes and the number ofganglion cells counted.


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48unilateral enucleates is about the same, but the percentage of unmyelinated axonsincreases relatively in optic nerves contralateral to a tract lesion.

By comparing the total number of axons in the optic nerve in animals whichreceived a tract lesion alone with that reported for the optic nerve of normal animals,it is apparent that only several thousand of the 117,000-130,000 fibers were left intactby the lesion. If the opposite eye was removed together with the tract lesion (Fig. 1 D),there seem to be more optic nerve axons remaining, although the total number ofuncrossed fibers in optic nerve remains small in comparison with that found in theunilateral enucleate (Experiment B, Table II). It is not clear why there is such a largedifference in the number of uncrossed fibers between animals which received a tractlesion and those in which one eye was removed, but a number of possible explanationscan be given. One possibility is that the tract lesion eliminated some of the uncrossed

CLT (L)

DLT 0%

1234567 1234637 1234337

Fig. 8. Comparison of cell counts and measurements in central regions (1,4,7,10 in Fig. 6), intermediateregions (2,5,8, 11 in Fig. 6) and peripheral regions (3,6,9, 12 in Fig. 6) in lower temporal quadrant ofretinas from same animals in which the pooled data is shown in Fig. 7. The abscissa and ordinate of eachof the histograms signify the sizes and the number of ganglion cells counted, respectively. Numbersl-7 represent ganglion cells of 6, 8.5, 11, 13.5, 16.5, 20 and 23 ,~m in somal diameter. Rand L signifyright and left retinas, whereas LT and LE signify left tract lesion and left eye enucleation made at birth.


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49axons which are collateral branches of the crossed axons injured by the lesion. Theresults of experiment D indicate that this is not true of all the axons, either becausethey do not all branch or for other reasons such as relative maturation at the time ofthe lesion. Another factor which may contribute to the higher fiber counts of un-crossed axons in the optic tract in the unilaterally enucleated animal is that some ofthe crossed fibers which arise from the opposite side of the brain or cross in Guddenscommissure3,17~1*,23,43,4s ight possibly join the optic tract where the uncrossed opticaxons reside. These fibers would be eliminated in animals which received a tract lesion.This has not been tested directly.Ganglion cell layer counts and measurements

While fiber counts are useful in determining the number of axons whichcontribute to the enlarged uncrossed pathway, ganglion cell layer counts andmeasurements can provide information regarding the effects of various experimentalmanipulations upon the number, types and distribution pattern of ganglion cells in theretina. As pointed out in Materials and Methods, cell counts and measurements weremade only in the lower temporal quadrant of the retina (Figs. 5 and 6) from which theuncrossed retinotectal pathway predominantly arises. The results were plotted ashistograms shown in Figure 7. Each of the 7 categories of cells in each of the 4histograms represents average data taken from 3 separate retinas. All data werenormalized according to the number of cells counted in the retina with the lowest totalnumber of cells counted in I2 fields, so that the results from different experiments canbe directly compared statistically (Students j-test). Although the non-normalized dataindicate that A and B, C and D are significantly different at 0.05 level in all categoriesbut the smallest (I), the difference among categories 3, 5, 6 and 7 was not significantafter the data were normalized. No detectable difference among the various categoriesof cells in A and C was observed. The average total number of ganglion cells countedin 12 fields in 3 retinas is indicated above the corresponding histogram while the totalnumber after normalization is shown in the parenthesis. Comparison of the non-normalized total number of cells between A and B, and that of C and D indicates thatless than half of the population of cells in the lower temporal quadrant of the retinawas eliminated by the tract lesion. The decrease of the larger cells and the increase ofthe small category 2 cells as shown in B and D suggests that many of the cells whichwere affected by the lesion were shrunken or in the process of degeneration. Fig. 8illustrates the effect of the lesion upon ganglion cells in various parts of the retinacontralateral to the tract lesion (Fig. 8B and D) in comparison with that observed inthe retina ipsilateral to the lesion and in the normal control rats (Fig. 8A and C). It isobvious that the effect of the tract lesion is much more severe in the central than in theperipheral retina where the number of cells in the ganglion cell layer contralateral tothe tract lesion and in the normal animal are 110,000 and 230,000, respectively. Thisfigure excludes a population of small darkly stained cells comprising 5-7 % of the totalpopulation. These are encountered throughout the whole area of the retina and areconsidered to be non-neuronal cells.


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Fig. 9. Degeneration pattern of the uncrossed retinotectal pathway from the right eye in a normal con-trol (A and C) and an animal in which the left optic tract is obliterated completely by a thalamic lesion(Band D). A and B are low power views of degeneration in the anteromedial portion of the right colli-culus. C and D are higher magnification views of regions in stratum opticumand B, respectively. Fink-Heimer. A and B x 125, C and D x 300. marked by a square in A


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Fig. 10. Degeneration pattern of the uncrossed retinotectai pathway from the right eye in animals whichreceived left eye enucleation (A and C) and a complete left optic tract lesion in addition to left eye enucle-ation (B and D) made at birth. A and B are low power views of degeneration in the anteromedial portionof the right colliculus. C and D are higher magnifications of the regions marked by a square in A and B,respectively. Fink-Heimer. A and B x 125, C and D x 300.


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Fig. 11. Degeneration pattern of the uncrossed retinotectal pathway from the right eye in animals whichreceived an incomplete left optic tract lesion (A and C), and left eye enucleation in addition to an in-complete left tract lesion (B and D) made at birth. A and B are low power views of degeneration in theanteromedial portion of the right colliculus. C and D are higher magnification views of the regionsmarked by a square in A and B, respectively. Fink-Heimer. A and B x 125, C and D x 300.


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53DISCUSSION

It is shown in the present study that enlargement of the terminal field of theuncrossed retinotectal pathway from one eye in the rat occurs after a contralateraltract lesion made at birth, and enlargement of the uncrossed pathway can be furtherenhanced by removal of the opposite eye. These findings are significant for a numberof reasons. First of all, it is known in the rat that at birth each of the eyes projectsbilaterally, but the uncrossed retinotectal pathway becomes progressively reduced overthe next few days unless the opposite eye is removed 24. The results reported here showthat the uncrossed pathway maintains and even enlarges its terminal field after acontralatelal lesion of the optic tract, suggesting that retraction of the uncrossedpathway depends not only on interaction between axonal terminals of the two eyes butalso on the integrity of crossed axons from that same eye.

A second point of interest lies in the comparison with results obtained in thedeveloping chick optic pathway 495. A transient ipsilateral isthmo-optic pathway hasbeen observed in the chick embryo during the second half of the incubation period. Ifthe opposite eye-cup which is the normal target of the isthmo-optic nucleus isremoved early in incubation, the number of isthmo-optic nucleus neurons which sendtheir axons to the ipsilateral side increases greatly. However, unlike the rats ipsilateralprojection which persists into adulthood, none of these cells survived beyond theperiod during which the normal transient uncrossed pathway is observed. In relationto the present results, it would be interesting to know what would be the effect in thechick of a unilateral removal of one eye and the ipsilateral isthmo-optic nucleus on theprojection from the remaining isthmo-optic nucleus.

Several possible explanations can be given as to why a tract lesion would lead toenlargement of the uncrossed pathway in the present experiments. One is that theprimary tract lesion induces retrograde degeneration in the opposite eye. Thisdegeneration may initiate a non-specific sprouting of the growing axons into theipsilateral optic tract in which there is little degeneration. A second possibility is thatthe changes observed after the tract lesion are a sprouting response which followsremoval of other pathways also damaged by the lesion. The only pathway projectingto the upper layers of the colliculus which would have been damaged by the lesion isthe relatively sparse crossed projection from the parabigeminal nucleus7. Otherpathways damaged would include the crossed projection arising in the colliculus andterminating in the nucleus lateralis posterior on the opposite side of the brain via thesupraoptic commissure, but this would not be relevant to the present results. A thirdpossibility is that the enlarged uncrossed pathway may be due to crossed axons still inthe process of growth which are directly rerouted at the optic chiasm to terminate onthe ipsilateral side of the brain. This seems to be reasonable because axons areprobably still growing out of the retina at the time when the tract lesion is made36. Themost likely explanation of the present results, perhaps, stems from the studies ofCunningham et al.s99,10 who showed that the axons of at least some retinal ganglioncells branch at the chiasm to innervate both sides of the brain in both normal rats andunilateral enucleates. In other words, the uncrossed axons are collateral branches of


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54the crossed axons. Therefore, cutting the crossed axons with a tract lesion apparentlywill prevent retraction of the uncrossed branches of these same axons. Since it is alsoshown in the present study that there is a reciprocal relation between the size of theuncrossed pathway and the size of the crossed projection left intact by an incompletetract lesion, results from the tract lesion experiments might suggest that if growth ofsome branches of axons is restricted, other branches of the same axons will enlargetheir terminal field.

These findings could be taken as support for Schneiders theory of conservationof terminal space1e,40,41 which implies at the cellular level that neurons can synthesizea limited amount of cytoplasm and/or make a limited number of synapses. Thesituation is not as simple as that, however, since there is considerable increase in thesize and extent of the uncrossed pathway if the tract lesion is accompanied by removalof the opposite eye, despite the finding that the number of uncrossed optic axons islittle increased compared with a tract lesion alone (see Table 11, C and D). Instead,these results indicate that the uncrossed axons are capable of enlarging their terminalfield in response to removal of competition from axons of the opposite eye. Evidenceagainst a strict interpretation of the theory of conservation of terminal space alsocomes from a number of other studies. For example, it has been shown in the rat thatthe uncrossed corticotectal pathway can be made to project bilaterally of the oppo-site cortex is removed in early postnatal days 25935. In addition, the aberrant cortico-tectal axons will further enlarge their territory by invading the superficialportion of the colliculus deafferented by eye enucleation22J5. Such a consecutiveincreased sprouting phenomenon following a cortical lesion and eye removal stronglysuggests that axons are potentially capable of increasing the size of their axonal arborswell beyond the normal limits. The results showing that there is an expansion inprojection after removal of other pathways strongly suggest that axonal interactionmust play a part in limiting enlargement of the terminal field of a fiber pathway.Frequently in the visual system such interaction appears to result in a segregation ofthe terminal areas served by each eye6,26,3.*,39,40,44. A similar limitation of terminalfields has also been demonstrated elsewhere in the central nervous system7132,33,47,48 aswell as in peripheral innervation patterns 1. It was hypothesized from the sensoryinnervation experiments that substances transported by neurons along their axons andreleased at the terminals function to limit the growth of adjacent neurites. The presentresults are consistent with such a proposal. Removal of an eye alone might remove amajor source of an inhibitory factor which would normally suppress or limit thegrowth of uncrossed optic axons. Tract section, on the other hand, might divert extrainhibitory material along the ipsilateral branch if optic axons do indeed branch tosupply both sides of the brain. This would result in the larger ipsilateral pathwayobserved in two-eyed animals with tract lesions.

The profound reduction in the size of the optic nerve contralateral to a tractlesion (see Table II, cases C and D) suggests that, if optic axons branch at the chiasm,either relatively few optic axons project bilaterally or the uncrossed branches areunable to sustain the neurons in the absence of the crossed branches. The large numberof cells resembling ganglion cells which persist, even when there are only 2OOO-3000


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55fibers in the optic nerve, suggests that as many as half of the cells in the ganglion celllayer may project internally, and perhaps are displaced amacrine cellsaJs. Thispopulation will severely bias any study using Nissl stains to define ganglion cell sizecategories in the rat retinal5 unless independent labeling criteria such as HRP aretaken into account.

In summary, the present results may be explained by invoking two generalprinciples. First, development of one part of an axonal arbor may occur at the expenseof other parts. This corresponds to Schneiders theory of conservation of terminalspace12,40. Secondly, inhibitory interactions between populations of axons are impor-tant in limiting the size of a terminal ramification. It is apparent here that rather than anatural limit being imposed on the size of a neurons axonal arbor by internal factorsalone, the external effects of interaction between axons are also important.ACKNOWLEDGEMENTS

We wish to thank Dr. R.P. Bolender for his valuable guidance in the statisticalanalysis and Ms. Cindy Gue for editorial assistance.

The research was supported by NIH Grants EY 00596, EY 01959 and EY 03414.

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(Land.), 234 (1973) 449-464.2 Bunt, A. H., Lund, R. D. and Lund, J. S., Retrograde axonal transport of horseradish peroxidase

by ganglion cells of albino rat retina, Bruin Research, 73 (1974) 215-228.3 Butler, A. B., Organization of ascending tectal projections in the lizard, Gekkogecko: a new patternof tectorotundal inputs, Bruin Research, 147 (1978) 353-361.4 Clarke, P. G. H. and Cowan, W. M., The development of the isthmo-optic tract in the chick withspecial reference to the occurrence and correction of developmental errors in the location and

connections of isthmo-optic neurons, J . camp. Neural., 167 (1976) 143-164.5 Clarke, P. G. H., Rogers, L. A. and Cowan, W. M., The time of origin and the pattern of survivalof neurons in the isthmo-optic nucleus of the chick, J . camp. Neural., 167 (1976) 125-142.6 Constantine-Paton, M. and Law, M. I., Eye-specific termination bands in tecta of three-eyed frogs,

Science, 202 (1978) 639-641.7 Cotman, C. W., Matthews, D. A., Taylor, D. and Lynch, G., Synaptic rearrangement in the dentategyrus : histochemical evidence of adjustments after lesions in immature and adult rats, Proc. nut.Acad. Sci. (Wash.), 70 (1973) 3473-3477.

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experimentally induced enlargement of the uncrossed retinotectal pathway in rats

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