Retinotopically inappropriate synapses of subnormal density formed by surgically misdirected optic fibers in goldfish tectum

304

Developmental Brain Res'ea~oh. ~8 (1988) 3(14--312

Ets( viol BRD 60250

Retinotopicalty intqlprq m syntCus of by surgi,,,4tl mi.dtrtcted in

density formed

William Pfir Hayes* and Ronald L. Meyer Department of Developmental and Cell Biology, Developmental Biology Center, University of Calijornia. lrvmc. ('A c)2717 ¢I,'. 5. A. i

(Accepted 10 November 1987) Key words: Topography; Competition; Regeneration; Fasciculation; Synaptogenesis, Horseradish pcroxidasc:

Electron microscopy: Synaptic density

Selected optic fibers were surgically deflected from one tectum onto the opposite host tectum which was denervatcd by eye cnucleation. At 6-8 months, deflected fibers were labeled with horseradish peroxidase and the retinotopically inappropriate part of rectum was examined using electron microscopy. Numerous (labeled) optic synapses were found in the primary optic innervation layer of the 'wrong' part of tectum but they were about half the normal density. The number and density of non-optic synapses was not found to be affected. These findings indicate optic fibers compete with each other but not with non-optic fibers for synaptic sites in tectum.

When the goldfish optic nerve is crushed, fibers regenerate to reform a retinotopic projection even though some fibers traverse incorrect routes 5A~'23"32. Direct surgical misrouting such as when optic fibers from one tectum are deflected into an inappropriate starting position into the opposite host tectum also result in reinnervation of appropriate regions 15't7"19 even when host optic fibers are absent 1~'21. Similarly, fibers selectively reinnervate tectum when part of retina has been surgically removed 3"2229'34. These findings and others (reviewed in refs. 7 and 8) provide strong support for the doctrine of neural specificity formulated by Sperry 3° that regenerating optic fibers selectively grow to the appropriate target cells and make synapses there. However, there is also experimental evidence for neural plasticity in this system 7'8. Optic fibers become compressed onto surgically formed half tectum such that retinotopic relations are maintained 4'14'25. Studies using half retinae and deflected optic fibers have also shown that fibers in these projections generally occupy a greater part of tectum than they would if they were part of complete optic projection 16'21'22"2934. To date, it is not known whether optic fibers which project to these in-

appropriate areas form synapses, and if so, how many. Autoradiography 16.2l2:'29 or horseradish peroxidase (HRP) 34 at the light microscopic level and presumed presynaptic electrophysiological recordings 1"2t'22'2~'3"~ used in previous studies only indicate the presence of optic fibers in the primary optic synaptic lamina (SFGS), not whether synapses are actually formed in these inappropriate areas. To address the question of whether inappropriate synapses are actually formed, a deflection protocol was used in which a subset of fibers was directed into the host tectum which was denervated of other optic fibers. The retinotopically inappropriate part of tecturn was then examined at the ultrastructural level (EM) after labeling optic fibers with HRP. A quantitative sampling method was used to also address the question of how many synapses form when the number of optic fibers is reduced and the projection is expanded across a larger than normal area of tectum. This was of particular interest because of previous H R P - E M studies that had shown that optic fibers regenerating into either an intact or a half tectum always formed normal numbers of synapses indicating that normal numbers were somehow constrained by

* Present address: NIH, CHHD, Building 36, Room 2A-21, Bethesda, MD 20892, U.S.A. Correspondence: R.L. Meyer, Developmental Biology Center, University of California, Irvine, Irvine, CA 92717, U.S.A

0165-3806/88/$03.50 I~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

305

tectum 11-t3'24'25. Would a similar constraint also operate in the innervated regions of tectum when the number of optic fibers was substantially less than normal? A preliminary report of these findings was previously made ~. Optic fibers were surgically deflected from one tectum onto the opposite host tectum which was simultaneously denervated by eye enucleation in goldfish 5 - 7 cm in body length. In 4 fish, optic fibers from the lateral brachia of the donor tectum were deflected, and in one fish, fibers from the medial brachia were deflected using surgical procedures described previously 21. Fibers were cut in a strip from surrounding tectum along the lateral or medial periphery and lifted over the midline and inserted into a small incision in anteromedial host tectum such that only fibers exiting the posterior part of the strip entered the host tectum. It has been previously shown by electrophysiological recordings that only fi-

bers normally innervating lateral posterior and medial posterior quadrants of tectum, respectively, are deflected by this procedure zl (see also refs. 21 and 33 for anatomical evidence). At 6 - 8 months, deflected optic fibers were labeled with H R P by applying a polyethylene tube filled with a concentrated solution of H R P to the cut optic nerve corresponding to the deflected fibers as detailed elsewherel°. Revived fish were then maintained at 5 - 7 °C for 48-60 h, and subsequently anesthetized and perfused with mixed aldehydes. One-hundred micron sections were cut through tectum at 10-12 °C with a vibratome. For electron microscopy the sections were reacted using the diaminobenzidine and cobalt ( D A B - C o ) intensification procedure I and then osmicated, dehydrated and embedded in epon-araldite. Material from 13 additional fish was reacted using para-phenylenediamine (PPD-PC) as the chromogen for light microscopy31.

Fig. 1. Light micrograph of HRP labeled optic fibers from lateral brachia in the anteromedial part of host tectum at two months regeneration. Note deflected fibers form large trunks (arrows) which give rise to smaller bundles and terminal-like arbors (arrowheads). Bar = 0.2 ram.

306 A t the light microscopic level, deflected optic fibers were observed forming unusually large bundles just posterior to the site of their insertion in anteromedial host tectum (Fig. 1). In fish with lateral brachial deflections, these bundles were observed to course dorsoventrally and to form branches which extended into the lateral and medial parts of tectum, These bundles gave rise to arbors which f o r m e d terminal-like regions in the anterior part of tectum ( Fig.

1 ). In agreement with prior studies using radioactive tracers l~''n, labeled optic fibers were confined to the primary optic fiber lamina (SO) and the two primary optic synaptic laminae, the thin superficial (S) lamina and d e e p e r thicker superficial fiber and gray lamina (SFGS). Consistent with the density of autoradiographic labeling observed previously~~ ~ the primary optic mnervation layer, the S - S O - S F G S , of host rectum was more sparsely labeled r e l a m c t o the labeling

Fig. 2. Electron micrograph photomontage of HRP labeled optic fibers from deflected lateral brachia in the S(_)and upper SFGS of anteromedial rectum at 8 months regeneration. Note optic fibers are grouped in unusually large fascicles and the large majority is myelinated. Note also the presence of labeled optic terminals in proximity to fascicles (arrowheads). Bar = 5 Lml.

Fig. 3. Electron micrographs of labeled optic terminals from deflected lateral brachial fibers making synaptic contacts in the SO and SFGS of anteromedial host rectum. A: labeled (optic) synaptic terminals (arrowheads) in proximity to an optic fascicle (arrows) in the non-optic SOl and optic SO where they are normally not observed (see text). B: higher magnification of the labeled SOi synapse in A. C: higher magnification of the labeled SO synapse in A. D: labeled optic terminal in the SFGS near labeled fascicle (arrows) making multiple (3) synaptic contacts (arrowheads) onto spine-like Type 1 postsynaptic profiles (see text). Bars in A = 2urn. B - D = 1 ,urn.

~a "-.4

308 found in the donor tectum. Deflected fibers were not detected in the deeper optic innervation laminae in the middle of central gray lamina (SGC) and at the border of the central white and periventricular laminae (SAC-SPV) in host tecta. The area selected for ultrastructural examination was the most heavily labeled area of anteromedial tectum at about 100-200 ~m posterior to the insertion site. Numerous large fascicles of labeled optic fibers (Fig. 2, 3A) and labeled optic terminals (Fig. 3 B - D ) were observed. The giant fascicles of myelinated optic fibers sometimes observed in or near the SO in fish with lateral deflections were larger and contained more fibers than observed previously in normal fish or in fish with regenerating optic nerves (Fig. 2). Smaller fascicles similar to those previously observed m were also observed in the SO (Fig. 3A) and SFGS. Labeled (optic) terminals were similar to those previously described as optic in goldfish using H R P 2"m'2a and degeneration 2"32. They often made multiple synaptic contacts which were asymmetric and typically were filled with synaptic vesicles (Fig. 3D). As observed previously m, they were often in proximity to or in contact with optic fascicles (Fig. 3 A - D ) . In contrast to normal fish and fish with regenerating optic fibers following nerve crush where optic fibers make synaptic contacts onto 4 classes of postsynaptic profiles n!, deflected optic fibers were observed forming synapses with only the most common class 1 postsynaptic profile which are spine-like. The distribution of labeled and unlabeled fiber and synapse numbers was determined from electron micrograph photomontages comprising uninterrupted sample columns through the H R P labeled S-SOSFGS (for details see ref. 11). This sampling method was used to avoid sampling errors caused by differences in the thickness in the innervation layer and to permit comparison between different fish with differing thickness of the optic laminae associated with various experimental manipulations J~-1324'25 Profiles were counted in bins that were 3 Mm deep and 14 Mm wide, and their numbers used to construct depth profiles using previously published criteria 1~. Depth profiles for each sample column were aligned using landmarks in the HRP labeled S-SO-SFGS. Depth profiles of mean numbers of labeled and unlabeled profiles for synapses (Fig. 4A) and fibers (Fig. 4D) were computed for fish with lateral brachial deflections at

6-8 months regeneration. These were compared lo mean depth profiles generated in a previous study ~ for normal fish with intact optic nerves (Fig. 4B, E) and fish 8 months after optic nerve crush (Fig. 4C, F). The depth profiles of synapses in fish with lateral deflections (Fig. 4A, D) showed a decreased density of optic synapses throughout the SFGS compared to normal fish (Fig. 4B, E) and fish (Fig. 4C, F) after long-term regeneration following nerve crush. In addition, there were fewer regenerating optic fibers in the SFGS of fish with lateral deflections than in fish after long-term regeneration although there were consistently more such fibers in the SO than in fish with complete (normal or regenerated) optic projections. Using the cytological criteria described previously 10"24'32, optic afferents from deflected lateral brachia appeared to be confined to the S-SO-SFGS as previously observed in normal and regenerating fish 10'11. However, the upper and lower boundaries were more difficult to define operationally as there were fewer labeled profiles. It was clear that the SSO-SFGS in fish with lateral brachial deflections wab thinner than found in fish with complete optic projections (see below). In addition, a laminar anomaly was observed in the organization of the superficial S-lamina. Normally optic fibers and synapses occupy this thin lamina which is just above or coincident with a peak in non-optic myelinated fibers (Fig. 4B, C, E, F). However, in fish with lateral brachial deflections the S-lamina was not clearly defined Instead labeled synapses were found in the intermediate optic fiber lamina, SOl (Figs. 3A, B, 4A, D), which lacks optic afferents in normal fish or in fish following nerve crush (Fig. 4B, C, E, F). The reduced thickness of the SFGS (see below) and anomalous organization of the S-lamina in these reduced optic projections suggests that the normal ultrastructural differentiation of the S-SO-SFGS may require a minimum number of optic fibers and synapses. The strict restriction of ~ptic fibers to the S-SO-SFGS was not aft'coted by fiber and synapse density and so is prestunably target-dependent. The depth profiles were used to compute mean total numbers of synapses and fibers per sample column through the S-SO-SFGS and the mean laminar thickness of the S-SO and SFGS ('Fable I). The syn* aptic counts were similar in all experimental fish and there was no substantial difference between lateral

309

Lateral Deflection

A 21

D

-

S

18

SO

SFGS

SGC

30~

S GS F

25

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!

12

16 9 10

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0

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6

9

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15

21

24

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~ 12

i

i

J

1

18

21

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9

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33

0

3

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tectal depth x 3p.m

S SO

18 15

g, 12

24

27

27

30

30

15

18

21

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33

tectal depth x 3p.m

Long-termRegeneration 21

21

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12

o~ 15

12

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0 -0 3

15

17.2 17.6

15

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12

tectal depth x 3p.m

Normal 21

9

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15 18 21 24 27 30 33 36 39 42 tectal depth x 3p.m

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Fig. 4. Mean depth profiles of optic and non-optic fiber and synapse n u m b e r s in the primary optic innervation layer, the S-SO-SFGS. A: optic (solid squares), non-optic (open squares), and total (crosses) synapse n u m b e r s from lateral brachial optic fibers a t . 6 - 8 months regeneration in anteromedial host tectum (n = 5 sample columns, 4 fish). B: synapse n u m b e r s f r o m normal fish with intact optic nerves (n = 5 columns, 3 fish; labels as in A). C: synapse n u m b e r s from fish with regenerating optic nerves at 8 m o n t h s regeneration (n = 4 columns, 2 fish; labels as in A). D: optic (solid squares) and non-optic (open squares) fiber n u m b e r s for columns in A. E: fiber numbers for columns in B (labels as in D). F: fiber n u m b e r s for columns in C (labels as in D).

310 TABLE 1 Summa; 3, o/sample column data Labeled synapses/ S-SO-SF(;S

Lateral deflection (n = 5, 4 fish) Medial deflection (n = 5.1 fish) Normal (n = 5, 3 fish}:~ Long-term (240 day) regeneration (n = 4, 2 fish)*

Unlabeled .~ynapses/ S-SO-SFGS

Total synapsese S-SO-SF(;S

44 ± 11

18(1 _+ 38

223 ± 46

6(I ±

53 _+ 1{~ 174_+ 15

144 ± 22 238±42

172 + _ 1(~

24(~ _+42

Labeled fibers' SFG5

Unlabeled fiber.s~ SF(;S

Laminar thickness (urn) .%-5¢)

,%1"(;S

26

2tl _+ 7

......

3S : t2

ItS7 + 38 411 i 3 3

59 ± 19 51 ±S

12 .+_3 24+8

19 ~_~ 22± ~

45 z: ~ 65 ± 5

41~ ~- 4~

223 4_ 70

33 ± 4

26 _~:5

77 ~:_~,

* Data from ref. 11.

or medial deflections. The n u m b e r of labeled synapses in fish with lateral deflections was decreased about 75% c o m p a r e d to fish with intact nerves or to fish with r e g e n e r a t e d nerves. The n u m b e r of optic fibers in the S F G S of fish with deflected optic fibers was decreased by 73% c o m p a r e d to fish after longterm regeneration. This was a c c o m p a n i e d by an apparent change in S - S O - S F G S thickness which was 30% less than that in normal fish and 41% less than in regenerated fish. Most of this decrease was attributable to a change in S F G S thickness (Table I). This decrease in the thickness of the S - S O - S F G S was only about half as much as the decrease in the n u m b e r of labeled synapses, and thus the average density of optic synapses in fish with deflected optic fibers was roughly half that found in fish with a full c o m p l e m e n t of optic fibers. F r o m the laminar analysis, it was evident that the decrease in synaptic numbers was expressed throughout the innervated region of the SFGS (Fig. 4 A - C ) . In contrast, the density of nonoptic synapses was roughly the same in fish with deflected optic fibers c o m p a r e d to normal fish or fish after long-term regeneration (Fig. 4 A - C ) . However, the total n u m b e r of unlabeled synapses per S-SOSFGS column was decreased by 2 4 - 2 7 % in deflected lateral brachial projections c o m p a r e d to complete optic projections (Table I) but this is most probably a consequence of the a p p a r e n t 31)-40% decrease in the thickness of the S - S O - S F G S . Thus, this decrease in the number of non-optic synapses should not be taken to mean that their n u m b e r undergo a real decrease in tectum since the lower b o u n d a r y of the S F G S was defined by the presence of labeled optic fibers in this

study. If optic fibers had simply failed to invade the lower 30f/~ of the actual SFGS and the number of non-optic synapses were corrected by a corresponding amount, then the n u m b e r of non-optic synapses would be normal. This interpretation would be in line with previous findings that net numbers of non-optic synapses do not change when optic innervation is eliminated -'a. However, the absence of good cytological landmarks at the S F G S - S G C boundary as noted previously m'2a warrants caution and leaves open the alternative possibility, namely, that the shrunken S F G S in our sample represents the real extent of this lamina and that there is a net loss of non-optic synapses within the SFGS. I11 either case, non-optic synapses apparently do not undergo reactive synaptogenesis (as shown in the m a m m a l i a n brain °) even after long-term optic deficits a finding that may in part account for the high degree of functional reinnervation in this system. The results in the present study provide direct evidence that retinotopically i n a p p r o p r i a t e synapses can form on rectum and this has significant implications for how specific synaptic connections are m a d e during map refinement and in the expression of various plasticities in this system. In m a p refinement, previous fiber tracing studies using W G A - H R P injected locally in the retina 23 or tectum 27 have shown that optic fibers initially project diffusely across about one-third of tectum and are then restricted to their a p p r o p r i a t e loci occupying about 10% of tecturn. During the early diffuse projection, it was previously found that the n u m b e r of optic synapses was normal and this n u m b e r r e m a i n e d constant thereaf-

311 ter 11. The present finding that optic fibers can form inappropriate synapses supports the interpretation that inappropriate synapses are formed during early regeneration and these are subsequently rearranged on an activity-dependent basis (see references in ref. 11). This was also indicated by the observation that T I ' X retinal blockade does not affect synapse number 12even though it inhibits map refinement 2°~2~. The formation of inappropriate synapses under the conditions of the present experiment can be explained by the absence of interactions with the complementary optic fibers. Previous studies have shown that when fibers are similarly deflected but host optic fibers are made to regenerate at the same time, deflected fibers are confined to their appropriate quadrant of tectum 17"19. Competition between optic fibers has been previously postulated to play a significant role in the formation of specific synaptic connections 8'26'35"36. The basis for this interaction may be competition for a limited number of postsynaptic sites on tectum. During optic nerve regeneration the normal number of optic synapses is established quite early, by 30 days, and remains constant thereafter l~ and optic fibers forced to regenerate onto surgically produced half tectum form normal numbers of optic synapses per tectal column, that is, each fiber forms, on average, half as many synapses on half rectum compared to intact tectum ~3"25. The present findings that the density of optic synapses is dramatically reduced when the number of fibers is decreased is consistent with this interpretation in that a substantial decrease in the number of fibers might be expected to decrease the competitive pressure and leave synaptic sites unfilled. If this competition is such that fibers are most competitive for their retinotopically appropriate part of rectum, then the decreased competitive conditions in the present experiment would lead to the formation of inappropriate synapses. Furthermore, the finding that the number and density of nonoptic synapses in the S-SO-SFGS is not affected by long-term optic deficit indicates optic fibers compete with each other but not with non-optic fibers for synaptic sites in tectum. The latter finding provides some additional insight

into the way in which synaptic numbers are regulated. It was not surprising that the total number of optic synapses would be less than normal since presumably there are metabolic limits on the number of synapses an individual fiber can support. However, there are two possible ways in which this could have occurred. One possible outcome was that optic fibers could have formed in normal density throughout their region of innervation until occupying as much tectal space as metabolically possible. In other words, fibers would saturate all local synaptic sites. The present results do not support this possibility in that all sample columns showed decreased density. The other possible result was for fibers to form synapses in decreased density, that is, for them to skip potential synaptic sites. This is apparently the case. It is important to note that decreased density was invariant in all sample columns and was observed in regions of tectum where the number of optic fibers was as great as that found in normal fish with intact nerves (though less than in fish with regenerating nerves); see Table I; Fig. 4 D - F . Thus, the result cannot be simply dismissed as a failure of fibers to invade the particular region sampled. A possible explanation for the subnormal synaptic density may be fiber-fiber repulsion such that optic fibers and synapses spread out by disaffinity s. However, this view seems inconsistent with the observation that deflected optic fibers form unusually large fascicles, and the large majority of normal and regenerating optic fibers are grouped into fascicles in tectum ~°'~ A more probable explanation is that deflected fibers are attracted by atrophic factor(s) or a synaptogenic factor(s) which is normally produced in limited quantity by tectal cells. When the number of competing fibers is reduced, this factor(s) would not be as scarce and the most effective way for fibers to maximize its uptake would be to disperse since this further reduces interfiber competition.

This work was supported by PHS Grants NS 16139 and EY 06746 to R.L.M. and PHS training Grant HD 07029 to W. P. H.

312 l Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (19811 775. 2 Airhart, M.J. and Kriebel, R.M., Retinal terminals in the goldfish optic tectum: identification and characterization, J. Comp. Neurol., 226 (1984) 377-390 3 Attardi, D.G. and Sperry, R.W.. Preferential selection of central pathways by regenerating optic fibers, Exp. Neurol., 7 (1963) 46-94. 4 Cook, J.E., Interactions between optic fibers controlling the locations of their terminals in the goldfish tectum, J. Embryol. Exp. Morphol., 52 (19791 89-103. 5 Cook, J.E., Tectal paths of regenerated optic axons in the goldfish: evidence from retrograde labeling with HRP, Exp. Brain Res., 51 (1983) 433-442, 6 Cotman, C,W. and Nieto-Sampedro, M., Cell biology of synaptic plasticity, Science, 225 (1984) 1287-1294. 7 Cowan, W.M. and Hunt, R.K.. The development of the retinotectal projection: an overview. In G.M. Edelman, W.E. Gall and W.M. Cowan rEds.), Molecular Bases of Neural Development, Wiley, New York, 1985, pp. 389-428. 8 Fraser, S.E., Cell interactions involved in neuronal patterning: an experimental and theoretical approach. In G.M. Edelman, W.E. Gall and W.M. Cowan rEds.), Molecular Bases of Neural Development, Wiley, New York. 1985, pp. 481-507. 9 Hayes, W.P. and Meyer, R.L., Inappropriate synapse formarion by misdirected regenerating optic fibers in goldfish: an electron microscopic horseradish peroxidase study, Soc. Neurosci. Abstr., 10 (1984) t036. 10 Hayes, W.P. and Meyer, R.L., Normal and regenerating optic fibers in goldfish tectum: HRP-EM evidence for rapid synaptogenesis and optic fiber-fiber affinity (submitted). 11 Hayes, W.P. and Meyer, R.L., Normal numbers of retinotectal synapses during the activity sensitive period of optic regeneration in goldfish: HRP-EM evidence implicating synapse rearrangement and collateral elimination during map refinement (submitted). 12 Hayes, W,P. and Meyer, R.L., Impulse blockade by intraocular tetrodotoxin during optic regeneration in goldfish: HRP-EM evidence that the formation of normal numbers of optic synapses and the elimination of exuberant optic fibers is activity independent (submitted). 13 Hayes, W.P. and Meyer. R.I_., Optic synapse number but not density is constrained during regeneration onto surgically-halved tectum in goldfish: HRP-EM evidence that optic fibers compete for fixed numbers of postsynaptic sites on rectum (submitted). 14 Horder, T.J. and Martin, K.A.C., Some determinants of optic terminal localization and retinotopic polarity within fiber populations in the tectum of goldfish, J. Physiol. (Lond.), 333 (1982) 481-5(!9. 15 Lo, R.Y.S. and Levine, R.L., Timecourse and pattern of optic fiber regeneration following tectal lobe removal in the goldfish, J. Comp. Neurol., 191 (1980)295-314. 16 Meyer, R.L., Deflection of selected optic fibers into a denervated rectum in goldfish, Brain Res., 155 (1978) 213-227. 17 Meyer, R.L., 'Extra' optic fibers exclude normal fibers from tectal regions in goldfish, J. Comp. Neurol.. 183 (1979) 883-901.

18 Meyer, R.L., Mapping the normal amt regenerating retin(> tectal projection of goldfish wifh autoradiographic methods, J. Cornp. Neurol., 189 (198n) 273-289. 19 Meyer, R.L., The growth and formation of ocular dominance columns by deflected optic fibers in goldfish. Dev. Brain Res., 6 (1983) 279-291. 20 Meyer, R.L., Tetrodotoxin inhibits the tormation ot re fined retinotopography in goldfish. Dev. Brain Re~., ~ ( 19831 293-298. 21 Meyer, R.L., Farget selection by surgically misdirectcc! optic fibers m the tectum of goldfish, Z Neurosci,, 4 (19841 235-250. 22 Meyer, R.L,, Tests for relabeling the goldfish rectum by oi> tic fibers, Dev. Brain Res., 31 (t987) 312 ~318 23 Meyer, R.[ .... Sakurai, K. and Schauwecker, E,, Topography of regenerating optic [ibers in goldfish traced with local wheat germ injections into retina: evidence for discontinuous microtopography in the retinotectal proiectiom J. Comp. Neurol.. 239 (1985) 27-43. 24 Murray, M. and Edwards, M.A., A quantitative study of the reinnervation of the goldfish optic tectum following optic nerve crush, J. Comp. Neurol.. 209 ( 19821363-373. 25 Murray, M., Sharma, S. and Edwards, M.A., Target regulation of synaptic number in the c~mpressed retinotectal projection of goldfish, J. Comp. NeuroL. 209 (19821 374--385 26 Prestige, M.C. and Willshaw, D.,I.. ~)n a role for competition in the formation of patterned neural connections, ProrL R. Soc. Lond. B.. 19(/(1975)77-,98, 27 Rankin, E . C . C and Cook, J.E., topographic refinement of the regenerating retinotectal projection of the goldfish in standard laboratory conditions: a quantitative W G A - H R P study, Exp, Brain Res., 63 (19861 409-420. 28 Schmidt, J.T, and Edwards, D.L., Activity sharpens the map during the regeneration of the rctinotectal projection in goldfish, Brain Res., 269 (19831 29-31L 29 Schmidt, J.g., Cicerone, C.M. and F,aster, S.S., Expansion ~)t the retinal projection to the tectum in goldfish: an dectrophysiological and anatomical study, J, (7omp. Near(d, 177 (1978) 257-278. 30 Sperry, R.W., Chemoaffinity in the orderly growth of nerve fiber patterns and connection~. Proc. Natl. Acad. Sci, U.S.A., 50 (1963) 7(/3-710. 31 Spreafico, R., Cheema, S., Ellis, I..C. and Rustioni, A., On 'Comparison of HRP visualization methods', ,L llislochem. E)'toehem., 30 (1982) 487- 488. 32 Sttlrmcr, C.A.O. and Easter, S.S., A comparison of the normal and regenerated retinotectal pathways of goldtish, ,I. Comp. Neurol., 223 (19841 57-7~ 33 Stiirmer. C.A.O. and Easter, S.S.. Rules ol order in the rctinotectal fascicles of goldfish, J ,~&urosci.. 4 (1984) 11145- 1051. 34 Udin, S.B. and Gaze, M., Expansion and retmotopic order in the goldfish retinotectal map alter large retinal lesions, Exp. Brain Res., 50 (1983) 347-352. 35 Whitelaw, V.A. and Cowan, J.D.. Specificity and plasticity of retinotectal connections: a computational model, J. Neurosci., i (1981) 1369-1387. 36 Willshaw, D.J. and v o n d e r Malsburg, C., How patterned connectmns can be set up by self-organization, Pro~ R. Soc. Lond. B.. 194 (19761 43t-445