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Further studies on the restoration of the contralateral retinotectal projection following regeneration of the optic nerve in the frog
BRAIN RESEARCH
183
F U R T H E R STUDIES ON T H E R E S T O R A T I O N OF T H E C O N T R A L A T E R A L R E T I N O T E C T A L P R O J E C T I O N F O L L O W I N G R E G E N E R A T I O N OF T H E OPTIC NERVE I N T H E F R O G
R. M. GAZE AND M. J. KEATING* Neurobiology Research Unit, Physiology Department, Edinburgh University, Edinburgh, Scotland (Great Britain)
(Accepted February 1st, 1970)
INTRODUCTION Normal visuomotor coordination in lower vertebrates appears to depend upon a precise retinotopic projection of fibres from the eye to the contralateral optic tectum. In fishes and amphibians normal visual function may return after section and regeneration of the optic nerve1,8,11,14, z0. The view that this recovery of visual function is associated with a restoration of the normal retinotopically organized retinotectal projection has received behaviouraP 5, anatomical 2 and electrophysiological support3,5,12. It seems that the regenerating optic nerve fibres select their appropriate terminations within the tectum and the mechanism by which they do this is of great interest. Sperry 17-19 has suggested that each retinal ganglion cell is, during development, cytochemically specified by a system of crossed gradients and a similar process of specification of tectal cells also takes place. Subsequent synaptic connection is believed to occur only between optic axons and tectal cells of corresponding cytochemical specificity. Further information as to the mode of formation of these selective connections may be provided by a study of the spatial and temporal sequence of optic fibre innervation of the tectum during regeneration of the optic nerve in the adult animal. Ideally this would involve repeated electrophysiological mapping of the retinotectal projection in one animal at various stages during the regeneration of the optic nerve. The mapping procedure is, however, so traumatic that it is, up to the present time, barely feasible to do this. An alternative method is to map the retinotectal projection in a"group of animals at various intervals after section of the optic nerve and in this way to provide a series of 'time-sections' through the process of regeneration. Such a study was performed by Gaze and JacobsonL They described a variety of patterns * Barry Stevens Memorial Fellow, Mental Health Research Fund. Brain Research, 21 (1970) 183-195
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R. M. GAZE AND M. J. KEATING
TABLE I TABLE OF EXPERIMENTAL RESULTS
L = section of left optic nerve. R = crush of right optic nerve.
Animal
Operative procedure
Operationrecording interval (days)
RON 1 RON 2 RON 3
L L L
48 49 63
RON RON RON RON RON RON RON RON
4 5 6 7 8 9 10 11
L R R L L L L R
63 40 27 30 47 36 130 84
RON RON RON RON
12 13 14 15
L R L R
133 67 56 96
RON RON RON RON RON RON RON RON RON RON RON RON RON
16 17 19 20 22 23 24 25 26 27 28 29 30
R R R L L L R R R L L R L
95 149 264 307 320 447 380 315 396 456 458 362 96
Result
Negative Pattern 1 Approximation to pattern 3 to both tecta Negative Pattern 3 Negative Negative Negative Negative Unclassifiable Semi-normal to part of rectum Pattern 2 ~ 3 Pattern 1 Unclassifiable Semi-normal to part of tectum Pattern 3 Pattern 3 Pattern 3 Pattern 4 Pattern 4 Pattern 4 Pattern 3 Pattern 2 -+ 3 Pattern 3 Pattern 4 Pattern 4 Pattern 3 Negative
o f the retinotectal p r o j e c ti o n , i n c o m p l e t e patterns being f o u n d in the earlier stages o f r e g e n e r a t i o n while in the later stages the patterns were m o r e complete. E v e n in late stages, h o w e v e r , a n o r m a l p a t t e r n was the e x c e p t i o n r a t h e r t h a n the rule in t h a t m a n y o f the animals showed a n o m a l o u s b u t o r g an i zed p r o j e c t i o n p a t t e r n s in a d d i t i o n to the restored c o n t r a l a t e r a l retinotectal projection. A f u r t h e r series o f animals has been e x a m i n e d an d the results are r e p o r t e d in this paper. In c o n j u n c t i o n with the results o b t a i n e d earlier by G a z e an d J a c o b s o n 7 a fairly c o h e r e n t picture emerges o f the stages o f restitution o f the retinotectal p r o j e c t i o n d u r i n g r e g e n e r a t i o n o f the optic nerve.
Brain Research, 21 (1970) 183-195
FROG OPTIC NERVE REGENERATION
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TABLE II TABLE OF EXPERIMENTAL RESULTS ARRANGED
CHRONOLOGICALLY
ACCORDING
TO THE INTERVAL IN DAYS
B E T W E E N T H E L E S I O N OF T H E O P T I C N E R V E A N D T H E T E R M I N A L R E C O R D I N G E X P E R I M E N T A N D A L S O A C C O R D I N G T O T H E T Y P E OF O P T I C N E R V E L E S I O N
Right optic nerve crush Animal
RON RON RON RON
Interval
Left optic nerve section Result
Animal
RON RON RON RON
6 5 13 11
27 40 67 84
RON 16 RON 15
95 96
RON 17 RON 19
149 264
Negative Pattern 3 Pattern 1 Semi-normal to part of tectum Pattern 3 Semi-normal to part of tectum Pattern 3 Pattern 3
RON RON RON RON
315 362 380 396
Pattern 2 -+ 3 Pattern 3 Pattern 3 Pattern 3
25 29 24 26
Interval
Result
7 9 8 1
30 36 47 48
Negative Negative Negative Negative
RON 2 RON 14
49 56
Pattern 1 Unclassifiable
RON 4 RON 3
63 63
Negative Approximation to pattern 3 to both tecta Negative Unclassifiable Pattern 2 -~ 3 Pattern 4 Pattern 4 Pattern 4 Pattern 4 Pattern 4
RON RON RON RON RON RON RON RON
30 10 12 20 22 23 27 28
96 130 133 307 320 447 456 458
METHODS The a n i m a l s used were Rana temporaria a n d Rana esculenta. I n a preliminary operation the left optic nerve was cut (16 a n i m a l s ) or the right optic nerve was crushed (12 animals). U n d e r ether anaesthesia the m u c o s a was reflected from the cartilage in the r o o f of the m o u t h a n d this cartilage was opened with a scalpel to expose the optic chiasma. The nerve lesion was made in each case close to, a n d j u s t distal to, the chiasma. The a n i m a l s were allowed to recover f r o m the operation a n d after a week were h a n d - f e d twice a week with pieces of chopped heart. After the intervals e n u m e r a t e d in T a b l e I the a n i m a l s were again anaesthetized a n d set up for electrophysiological m a p p i n g of the visual projections. The details of the recording t e c h n i q u e have been described by Gaze a n d J a c o b s o n L I n the present investigation the microelectrodes used were metal-filled glass micropipettes tipped with p l a t i n u m . RESULTS The projection f r o m the eye to the contralateral optic t e c t u m was m a p p e d in 28 a n i m a l s in which either the left optic nerve h a d been cut or the right optic nerve h a d been crushed. The results are s u m m a r i s e d in Tables I a n d II. Seven a n i m a l s Brain Researeh, 21 (1970) 183-195
186
R. M. GAZE AND M. J. KEATING
27
10
25
29
39
31
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22
23
24
25
32
16
17
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O
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O
11
12
13
14
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5
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Fig. 1. The projection of the left visual field upon the right optic tectum in frog RON 2. The upper diagram represents the outline of the optic tectum seen from above. Each number on the tectum represents an electrode position at which an optimal response was produced when the stimulus was at the position indicated by the same number in the visual field. The perimetric chart extends radially for 100° from the centre of the visual field which is the 'fixation point' of the frog's left eye. N, S, T, I represent the nasal, superior, temporal and inferior poles of the left visual field. Similar conventions are used in the succeeding figures.
gave no responses f r o m the c o n t r a l a t e r a l t e c t u m ; in 6 o f these less than 50 d a y s h a d elapsed b e t w e e n the o p e r a t i o n on the optic nerve a n d the r e c o r d i n g e x p e r i m e n t , a n d in the 7th a n i m a l the i n t e r v a l was 96 days. A f u r t h e r 2 a n i m a l s gave unclassifiable results in t h a t the r e g e n e r a t e d r e t i n o t e c t a l p r o j e c t i o n arose f r o m diffuse areas o f the visual field a n d no o b v i o u s o r d e r c o u l d be f o u n d in the p r o j e c t i o n . T h e r e m a i n i n g 19 a n i m a l s c o u l d be classified a c c o r d i n g to the degree o f n o r m a l i t y o f the regenerated retinotectal projection. T h e t y p e o f p r o j e c t i o n p a t t e r n described b y G a z e a n d J a c o b s o n 7 as p a t t e r n 1 was seen in 2 animals (Fig. 1, R O N 2). The c h a r a c t e r i s t i c f e a t u r e o f this p a t t e r n o f r e g e n e r a t i o n is t h a t a relatively small r e g i o n o f the retina projects to a c o n s i d e r a b l y larger p a r t o f the d o r s a l tectal surface t h a n is the case in a n o r m a l animal. T h e rem a i n d e r o f the r e t i n a does n o t a p p e a r to h a v e c o n n e c t e d with the t e c t u m . W i t h i n t h e r e g i o n o f r e t i n a t h a t has re-established c o n n e c t i o n with the t e c t u m there is no o b v i o u s o r d e r in the p r o j e c t i o n o f retinal p o i n t s t o the tectum. As in the earlier e x p e r i m e n t s o f G a z e a n d J a c o b s o n 7, the a n i m a l s showing p a t t e r n 1 were r e c o r d e d s o o n after the Brain Research, 21 (1970) 183-195
187
FROG OPTIC NERVE REGENERATION
S
I
Fig. 2. The projection of the left visual field upon the right optic tectum in frog R O N 12
optic nerve lesion. Table II shows that these animals occur early in the experimental time-series. A second type of incomplete restoration of the retinotectal projection, in which the projection was appropriately organized across one tectal axis but not across the other, was described by Gaze and Jacobson 7 as pattern 2. A projection similar to this in type was seen in 2 animals in the present series and Fig. 2 (RON 12) illustrates one of these. In pattern 2 the greater part of the tectal projection arises, as in pattern 1, from only a small region of the retina, but in addition the projection to the rostral tectum from the nasosuperior quadrant of the field (temporo-inferior retina) shows evidence of organization along the mediolateral axis of the tectum but not along its rostrocaudal axis. The projection shown in Fig. 2 is more organized than the pattern 2 projection illustrated by Gaze and Jacobson 7 (Fig. 5); in the present case not only is the projection to the most rostral tectum organized in the mediolateral axis of the tectum, but similarly even within the thin band of retina projecting to the greater part of the tectum there is appropriate organization across the mediolateral tectal axis (dorsoventral axis of field), although organization in the rostrocaudal tectal axis (nasotemporal field axis) is absent. A regenerated retinotectal projection showing a pattern intermediate between pattern 2 and the normal is shown in Fig. 3 (RON 25). In this animal there is still a portion of the retina, corresponding to the superior part of the visual field, which Brain Research, 21 (1970) 183-195
188
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R. M. GAZE AND M. J, KEATING
lITTI°TuM7 16
15
12 I
7
14
13
II
10
6
5
T
9
N
T I
Fig. 3. The projection of the right visual field upon the left optic tectum in frog RON 25. The unringed numbers on the perimetric chart represent a series of stimulus positions corresponding to the normal projection of the right visual field upon the left tectum. The ringed numbers represent additionall localized but anomalous stimulus positions projecting to the left tectum.
projects rather diffusely to the greater part of the tectum; but superimposed on this diffuse projection is an approximation to a normal retinotectal projection, organized appropriately in both tectal axes. In 8 animals there was, following regeneration of the optic nerve, a restoration of the normal retinotectal projection (Fig. 4, R O N 5). This type of regenerated projection was termed pattern 3 by Gaze and Jacobson 7. Pattern 4, as described by these authors, consisted of an essentially normal retinotectal projection with, in addition, arising from the nasosuperior periphery of the visual field, a retinotopically organized but anomalous projection to the rostral part of the contralateral rectum. In the present experiments this pattern was found in 5 animals, one of which is illustrated in Fig. 5 ( R O N 20). It m a y be seen that electrode positions in the rostral half of the contralateral tectum received input f r o m two positions in the visual field of one eye; one of these positions is that which normally projects to the contralateral tectum while the other, arising f r o m the nasosuperior periphery of the field (ringed numbers), comprises, with its fellows, a retinotopically Brain Research, 21 (1970) 183-195
189
FROG OPTIC NERVE REGENERATION
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35
34
33
32
31
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29
28
27
26
25
24
23
22
21
20
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Fig. 4. The projection of the right visual field upon the left optic tectum in frog R O N 5.
organized projection which is reversed along the nasotemporal field axis when compared with the normal contralateral projection. Thus in the normal projection (which is approximated by pattern 3 ; Fig. 4) as the electrode positions move caudally on the tectum the corresponding stimulus positions in the visual field move more temporally; but in the anomalous projection as the electrode positions move caudally, the stimulus positions in the field move nasally. Pattern 4 was only found after nerve section, never after nerve crush. All 5 animals showing pattern 4 were mapped a considerable time after the nerve lesion was made; Table [I shows that these patterns all occurred late in the experimental time-series. In all 5 animals in which pattern 4 was found, an anomalous visual projection was also found from the eye to the ipsilateral tectum (Fig. 6, R O N 20). In these animals there was reconstitution of a normal ipsilateral projection from the nasosuperior visual field to the rostral part of the ipsilateral tectum; there was also, however, an additional anomalous but retinotopically organized projection from the whole visual field to the ipsilateral tectum. In its organization this anomalous ipsilateral projection resembles the normal contralateral projection, with nasal field projecting to rostral rectum and temporal field to caudal tectum. Brain Research, 21 (1970) 183-195
190
R. M. GAZE AND M. J. KEATING
RIGHT
TECTUM
17
18
19
20
12
13
14
15
16
8
9
10
11
4
5
6
7
N
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Fig. 5. The projection of the left visual field upon the right optic tectum in frog RON 20. The unringed numbers on the perimetric chart represent the normal contralateral field projection. The ringed numbers constitute an additional series of stimulus positions to the right tectum and forming an additional but anomalous retinotopic projection.
In 1 o f the 5 animals with pattern 4 regeneration ( R O N 28) the pattern o f regeneration to the two tecta was dissimilar. The projection f r o m the left eye to the right tectum consisted o f a mirror-image ipsilateral projection together with a contralateral projection o f pattern 1 type. In this animal the projection f r o m the left eye to the left tectum was straightforwardly pattern 4; that is, there was a full but a n o m a lous contralateral projection to this t e c t u m together with a restored n o r m a l ipsilateral projection. DISCUSSION F o r reasons discussed by M a t u r a n a e t al. 13 and Gaze and J a c o b s o n 7 we believe that normally when recording f r o m the superficial layers o f the contralateral optic tectum we are recording activity f r o m the terminal arborizations o f optic nerve fibres. The m a p p i n g technique used thus indicates the distribution o f optic fibre terminals over the surface o f the tectum at various periods during regeneration o f the optic nerve. Brain Research, 21 (1970) 183-195
FROG OPTIC NERVE REGENERATION
191
s
7
16
I
Fig. 6. The projection of the left visual field upon the left optic tectum in frog RON 20. In this case the ringed numbers on the perimetric chart represent the normal field projection to the ipsilateral tectum whereas the unringed numbers constitute another series of stimulus positions also projecting to the left tectum and forming an additional but anomalous retinotopic projection.
The present results confirm and extend the observations of Gaze and Jacobson v. Negative results preponderate a m o n g those experiments with the shortest o p e r a t i o n recording intervals. The mean interval for those animals which gave no results was 49 days. Presumably the terminal mapping experiment t o o k place before the regenerating optic fibres had reached the rectum. Pattern 1 regeneration tends to be found after short operation-recording intervals. Since the original description of this pattern of early regeneration 7 evidence of similar 'spreading' of developing or regenerating optic nerve fibres over the tectum has been sought unsuccessfully in other species. The failure to demonstrate this spreading at the tectal level by means of classical histological methods in goldfish 2 or embryonic chicks 4 is not too surprising since the terminal fibres may be very small and dispersed and may hardly be traced with ordinary silver stains. Electrophysiological studies in fish 1°,zl have, however, also failed to demonstrate any spreading of optic nerve fibres. The two animals showing pattern 1 regeneration in this series confirm, however, that in the frog, during early stages of regeneration of the optic nerve, fibres f r o m Brain Research, 21 (1970) 183-195
192
It, M. G A Z E A N D M. J. K E A T I N G
small areas of the retina may spread over considerably greater areas of tectum than those to which they normally project. The work of Cronly-Dillon 3 on the regeneration of retinotectal connections in newts suggests that a comparable phenomenon may occur in urodeles. This author found that the multi-unit receptive field size of a given tectal locus was larger than normal during the earlier stages of regeneration; and this was particularly so in the case of projections from the peripheral regions of the retina. On the other hand the fact that in urodeles the entire retina degenerates and then regenerates after optic nerve lesions complicates interpretation of the results in these animals and, as Gaze and Watson 9 have shown, the regenerated central retina in newts is older than the regenerated peripheral retina. Gaze and Jacobson 7 suggested that the various patterns of regeneration, in numerical order, represented sequential stages in the restoration of the retinotectal projection. Our results would support this idea on two counts: firstly the time distribution of the various patterns in the present experiments suggests that pattern 1 is an early, and pattern 3 or 4 a late, manifestation of the regeneration process; and secondly the 2 animals classed as pattern 2 in the present series in fact represent intermediate stages between patterns 2 and 3. If the pattern being described represents successive stages in the process of regeneration, then the progression from pattern 1 to pattern 2 and thence to normal (pattern 3) indicates that, following the initial diffuse projection from small retinal areas, as more fibres regenerate to the tectum, the organization of these fibres occurs in stages. The first semblance of organization occurs at the rostral pole of the tectum and the organization then spreads over the rest of the tectum. This rostrocaudal tectal sequence of organization parallels the rostrocaudal sequence of stratification and acetylcholinesterase appearance during development and in both cases the rostrocaudal direction of spread is presumably related to the fact that the optic axons grow in from the rostral end of the tectum. It would be interesting in this context to observe the result of growing an optic nerve into the tectum from an abnormal direction. It seems likely that during the process of regeneration of the optic fibres order arises out of the initially diffuse early patterns due to a process of competition for appropriate tectal positions by the regenerating fibres. Thus the first fibres to regenerate spread diffusely over the tectum since there are no other fibres with which to compete. As larger numbers of fibres arrive competition increases and aberrant connections will be displaced as fibres more appropriate to the tectal locus arrive. Large numbers of regenerating fibres will arrive first at the rostral pole of the tectum and thus semblances of organization appear here first. Direct evidence for the competitive re-innervation of tectal loci is provided by Gaze and Sharma 8 and a similar process of competition is suggested by Cronly-Dillon a. It is not immediately apparent, however, why the regenerating fibres become organized along the mediolateral axis of the tectum (dorsoventral field axis) before they do along the rostrocaudal tectal axis (nasotemporal field axis). It is conceivable that this axial difference reflects the mode of ingrowth of regenerating fibres. If Brain Research, 21 (1970) 183-195
FROG OPTIC NERVE REGENERATION
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OPTIC
Fig. 7. See text.
we conceive of the main mass of fibres advancing rostrocaudally over a wide front and these fibres have branched, then along any mediolateral strip of rostral tectum there will be a chance of fibre competition occurring before it can occur in the rostrocaudal axis. In this case the axial differences noted would reflect the temporal sequence and direction of innervation. Many fibres arriving more or less simultaneously at level AB (mediolateral axis, Fig. 7) will be able to start the sorting process; whereas sorting along the rostrocaudal axis (Fig. 7 BC) cannot yet start, since the main fibre mass has not yet reached C. Fibre competition and tectal axial differences are further considered by Gaze and Sharma s. Eight animals showed a normal retinotectal projection following regeneration of the optic nerve. In these cases the operation-recording interval varied from 40 to 396 days (Tables I and II). Five animals gave the intriguing pattern 4. This pattern consists, in a way, of a 'supernormal' projection, in that in addition to the restored normal retinal projection to the contralateral rectum there is a retinotopically organized projection from the nasosuperior periphery o f the field to the rostral tectum (Fig. 5). This anomalous contralateral projection is in fact an ipsilateral projection which has appeared in the 'wrong' tectum 6,v. More will be said about the significance of the anomalous pattern 4 projection in a subsequent paper on the restoration of the ipsilateral visual projection following regeneration of the optic nerve. Here we need say only that the basic abnormality in this type of regeneration pattern is that the regenerating nerve fibres appear to regenerate directly not only to the contralateral tectum but also to the ipsilateral tectum; and they thus produce mirror-image contralateral-type projections in both tecta (Figs. 5 and 6). It was suggested by Gaze and Jacobson v that pattern 4 may arise as a progression from pattern 3 in that the former is a 'super-complete' type of regeneration. However, a consideration of Table II indicates that patterns 3 and 4 are probably not sequential stages of regeneration but are alternative endpoints of the regeneration process, dependent upon the type of operation used to produce the lesion in the optic nerve. Crush lesions, which leave the sheath of the nerve intact, lead to pattern 3; nerve sections, which permit aberrant fibres to enter the diencephalon ipsilaterally, lead Brain Research, 21 (1970) 183-195
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R. M. GAZE AND M. J. KEATING
t o p a t t e r n 4. T h e ability o f r e t i n a l axons t o f o r m a p p r o p r i a t e c o n n e c t i o n s with either tectum, i f given the o p p o r t u n i t y , was i l l u s t r a t e d b y S p e r r y 16 w h o d e l i b e r a t e l y crossu n i t e d o p t i c nerves to the i p s i l a t e r a l optic t r a c t . SUMMARY
T h e c o n t r a l a t e r a l r e t i n o t e c t a l p r o j e c t i o n was m a p p e d in a series o f frogs at v a r i o u s intervals after i n t e r r u p t i o n o f the optic nerve. F u r t h e r evidence o f a n early diffuse stage o f r e g e n e r a t i o n has been o b t a i n e d . F o l l o w i n g this diffuse stage there a p p e a r s to o c c u r an o r g a n i z a t i o n o f the tectal c o n n e c t i o n s o f o p t i c fibres initially a l o n g the m e d i o l a t e r a l axis o f the t e c t u m a n d subsequently a l o n g the r o s t r o c a u d a l tectal axis. Some m o r e i n t e r m e d i a t e p a t t e r n s o f r e g e n e r a t i o n h a v e been d e s c r i b e d s u p p o r t i n g the view t h a t these p a t t e r n s are stages in a c o n t i n u o u s sequence. T h e occurrence o f p a t t e r n 4 r e g e n e r a t i o n to t h e c o n t r a l a t e r a l t e c t u m has been s h o w n to be a s s o c i a t e d i n v a r i a b l y with a n o m a l o u s r e g e n e r a t i o n to the i p s i l a t e r a l tectum. It is suggested t h a t p a t t e r n 3 a n d p a t t e r n 4 are a l t e r n a t i v e e n d - p o i n t s o f the r e g e n e r a t i o n sequence, d e p e n d e n t u p o n the t y p e o f o p e r a t i v e p r o c e d u r e on the optic nerve. ACKNOWLEDGEMENT
W e are grateful t o M i s s E. F o r r e s t f o r h e r excellent h i s t o l o g i c a l assistance.
REFERENCES 1 ARORA, H. L., AND SPERRY, R. W., Color discrimination after optic nerve regeneration in the fish
Astronotus ocellatus, Developm. Biol., 7 (1963) 234-243. 2 ATTARDI, D. G., AND SPERRY, R. W., Preferential selection of central pathways by regenerating
optic fibres, Exp. Neurol., 7 (1963) 46-64. 3 CRONLY-DILLON, J., Pattern of retinotectal connections after retinal regeneration, J. Neurophysiol., 31 (1968) 410-418. 4 DE LONG, R. G., AND COULOMBRE,A. J., Development of the retinotectal topographic projection in the chick embryo, Exp. Neurol., 13 (1965) 351-363. 5 GAZE, R. M., Regeneration of the optic nerve in Xenopus laevis, Quart. J. exp. Physiol., 44 (1959) 290-308. 6 GAZE, R. M., AND JACOBSON, M., The projection of the binocular visual field upon the optic tecta of the frog, Quart. J. exp. Physiol., 47 (1962) 273-280. 7 GAZE, R. M., AND JACOBSON,M., A study of the retinotectal projection during regeneration of the optic nerve in the frog, Proc. roy. Soc. B, 157 (1963) 420-448. 8 GAZE, R. M., AND SHARMA, S. C., Axial differences in the reinnervation of the goldfish optic tectum by regenerating optic nerve fibres, Exp. Brain Res., 10 (1970) 171-181. 9 GAZE, R. M., AND WATSON, W. E., Cell division and migration in the brain after optic nerve lesions. In G. E. W. WOLSTENHOLMEAND M. O'CONNOR (Eds.), Growth of the Nervous System, Ciba Symposium, Churchill, London, 1968, pp. 53-67. 10 JACOBSON, M., AND GAZE, R. M., Selection of appropriate tectal connections by regenerating optic nerve fibres in adult goldfish, Exp. Neurol., 13 (1965) 418-430. 11 MATTHEY,R., RGcuperation de la vue aprGs rGsection des nerfs optiques chez le Triton, C. R. Soc. Biol. (Paris), 93 (1925) 904-906. 12 MATURANA,H. R., LETTVIN,J. Y., McCULLOCH,W. S., AND PITTS, W. H., Physiological evidence that cut optic nerve fibres in the frog regenerate to their proper places in the tectum, Science, 130 (1959) 1709-1710. Brahz Research, 21 (1970) 183-195
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13 MATURANA,H. R., LFTTVIN, J. Y., McCULLOCH, W. S., AND PtTTS, W. H., Anatomy and physiology of vision in the frog, J. gen. Physiok, 43, Suppl. (1960) 129-175. 14 SPERRY,R. W., Visuomotor coordination in the newt (Triturus viridescens) after regeneration of the optic nerve, J. cutup. Neurok, 79 (1943) 33-55. 15 SPERRY, R. W., Optic nerve regeneration with return of vision in anurans, J. Neurophysiol., 7 (1944) 57-70. 16 SPERRY,R. W., Restoration of vision after crossing of optic nerves and after contralateral transplantation of the eye, J. Neurophysiol., 8 (1945) 15-28. 17 SPERRY,R. W., Mechanisms of neural maturation. In S. S. STEVENS(Ed.), Handbook of Experimental Psychology, Wiley, New York, 1951, pp. 236-280. 18 SPERRY,R. W., Chemoaffinity in the orderly growth of nerve fibre patterns and connections. Proc. nat. Acad. Sci. (Wash.), 50 (1963) 703-710. 19 SPERRY, R. W., Embryogenesis of behavioural nerve nets. In R. L. DE HAAN AND H. URSPRUNG (Eds.), Organogenesis, Holt, Rinehart and Winston, New York, 1965, pp. 161-186. 20 WEILER, I. J., Restoration of visual acuity after optic nerve section and regeneration in Astronotus ocellatus, Exp. Neurol., 15 (1966) 377-386. 21 WESTERMAN,R. k., Specificity in regeneration of optic and olfactory pathways in teleost fish. In D. R. CURTIS AND A. K. MClNTYRE (Eds.), Studies in Physiology, Springer, Berlin, 1965, pp. 263-269.
Brain Research, 21 (1970) 183-195
183
F U R T H E R STUDIES ON T H E R E S T O R A T I O N OF T H E C O N T R A L A T E R A L R E T I N O T E C T A L P R O J E C T I O N F O L L O W I N G R E G E N E R A T I O N OF T H E OPTIC NERVE I N T H E F R O G
R. M. GAZE AND M. J. KEATING* Neurobiology Research Unit, Physiology Department, Edinburgh University, Edinburgh, Scotland (Great Britain)
(Accepted February 1st, 1970)
INTRODUCTION Normal visuomotor coordination in lower vertebrates appears to depend upon a precise retinotopic projection of fibres from the eye to the contralateral optic tectum. In fishes and amphibians normal visual function may return after section and regeneration of the optic nerve1,8,11,14, z0. The view that this recovery of visual function is associated with a restoration of the normal retinotopically organized retinotectal projection has received behaviouraP 5, anatomical 2 and electrophysiological support3,5,12. It seems that the regenerating optic nerve fibres select their appropriate terminations within the tectum and the mechanism by which they do this is of great interest. Sperry 17-19 has suggested that each retinal ganglion cell is, during development, cytochemically specified by a system of crossed gradients and a similar process of specification of tectal cells also takes place. Subsequent synaptic connection is believed to occur only between optic axons and tectal cells of corresponding cytochemical specificity. Further information as to the mode of formation of these selective connections may be provided by a study of the spatial and temporal sequence of optic fibre innervation of the tectum during regeneration of the optic nerve in the adult animal. Ideally this would involve repeated electrophysiological mapping of the retinotectal projection in one animal at various stages during the regeneration of the optic nerve. The mapping procedure is, however, so traumatic that it is, up to the present time, barely feasible to do this. An alternative method is to map the retinotectal projection in a"group of animals at various intervals after section of the optic nerve and in this way to provide a series of 'time-sections' through the process of regeneration. Such a study was performed by Gaze and JacobsonL They described a variety of patterns * Barry Stevens Memorial Fellow, Mental Health Research Fund. Brain Research, 21 (1970) 183-195
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R. M. GAZE AND M. J. KEATING
TABLE I TABLE OF EXPERIMENTAL RESULTS
L = section of left optic nerve. R = crush of right optic nerve.
Animal
Operative procedure
Operationrecording interval (days)
RON 1 RON 2 RON 3
L L L
48 49 63
RON RON RON RON RON RON RON RON
4 5 6 7 8 9 10 11
L R R L L L L R
63 40 27 30 47 36 130 84
RON RON RON RON
12 13 14 15
L R L R
133 67 56 96
RON RON RON RON RON RON RON RON RON RON RON RON RON
16 17 19 20 22 23 24 25 26 27 28 29 30
R R R L L L R R R L L R L
95 149 264 307 320 447 380 315 396 456 458 362 96
Result
Negative Pattern 1 Approximation to pattern 3 to both tecta Negative Pattern 3 Negative Negative Negative Negative Unclassifiable Semi-normal to part of rectum Pattern 2 ~ 3 Pattern 1 Unclassifiable Semi-normal to part of tectum Pattern 3 Pattern 3 Pattern 3 Pattern 4 Pattern 4 Pattern 4 Pattern 3 Pattern 2 -+ 3 Pattern 3 Pattern 4 Pattern 4 Pattern 3 Negative
o f the retinotectal p r o j e c ti o n , i n c o m p l e t e patterns being f o u n d in the earlier stages o f r e g e n e r a t i o n while in the later stages the patterns were m o r e complete. E v e n in late stages, h o w e v e r , a n o r m a l p a t t e r n was the e x c e p t i o n r a t h e r t h a n the rule in t h a t m a n y o f the animals showed a n o m a l o u s b u t o r g an i zed p r o j e c t i o n p a t t e r n s in a d d i t i o n to the restored c o n t r a l a t e r a l retinotectal projection. A f u r t h e r series o f animals has been e x a m i n e d an d the results are r e p o r t e d in this paper. In c o n j u n c t i o n with the results o b t a i n e d earlier by G a z e an d J a c o b s o n 7 a fairly c o h e r e n t picture emerges o f the stages o f restitution o f the retinotectal p r o j e c t i o n d u r i n g r e g e n e r a t i o n o f the optic nerve.
Brain Research, 21 (1970) 183-195
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TABLE II TABLE OF EXPERIMENTAL RESULTS ARRANGED
CHRONOLOGICALLY
ACCORDING
TO THE INTERVAL IN DAYS
B E T W E E N T H E L E S I O N OF T H E O P T I C N E R V E A N D T H E T E R M I N A L R E C O R D I N G E X P E R I M E N T A N D A L S O A C C O R D I N G T O T H E T Y P E OF O P T I C N E R V E L E S I O N
Right optic nerve crush Animal
RON RON RON RON
Interval
Left optic nerve section Result
Animal
RON RON RON RON
6 5 13 11
27 40 67 84
RON 16 RON 15
95 96
RON 17 RON 19
149 264
Negative Pattern 3 Pattern 1 Semi-normal to part of tectum Pattern 3 Semi-normal to part of tectum Pattern 3 Pattern 3
RON RON RON RON
315 362 380 396
Pattern 2 -+ 3 Pattern 3 Pattern 3 Pattern 3
25 29 24 26
Interval
Result
7 9 8 1
30 36 47 48
Negative Negative Negative Negative
RON 2 RON 14
49 56
Pattern 1 Unclassifiable
RON 4 RON 3
63 63
Negative Approximation to pattern 3 to both tecta Negative Unclassifiable Pattern 2 -~ 3 Pattern 4 Pattern 4 Pattern 4 Pattern 4 Pattern 4
RON RON RON RON RON RON RON RON
30 10 12 20 22 23 27 28
96 130 133 307 320 447 456 458
METHODS The a n i m a l s used were Rana temporaria a n d Rana esculenta. I n a preliminary operation the left optic nerve was cut (16 a n i m a l s ) or the right optic nerve was crushed (12 animals). U n d e r ether anaesthesia the m u c o s a was reflected from the cartilage in the r o o f of the m o u t h a n d this cartilage was opened with a scalpel to expose the optic chiasma. The nerve lesion was made in each case close to, a n d j u s t distal to, the chiasma. The a n i m a l s were allowed to recover f r o m the operation a n d after a week were h a n d - f e d twice a week with pieces of chopped heart. After the intervals e n u m e r a t e d in T a b l e I the a n i m a l s were again anaesthetized a n d set up for electrophysiological m a p p i n g of the visual projections. The details of the recording t e c h n i q u e have been described by Gaze a n d J a c o b s o n L I n the present investigation the microelectrodes used were metal-filled glass micropipettes tipped with p l a t i n u m . RESULTS The projection f r o m the eye to the contralateral optic t e c t u m was m a p p e d in 28 a n i m a l s in which either the left optic nerve h a d been cut or the right optic nerve h a d been crushed. The results are s u m m a r i s e d in Tables I a n d II. Seven a n i m a l s Brain Researeh, 21 (1970) 183-195
186
R. M. GAZE AND M. J. KEATING
27
10
25
29
39
31
O
O
22
23
24
25
32
16
17
1|
I!
O
/0
O
11
12
13
14
15
4
o
5
i
7
I
$I1
21
$
17 33 0 ~,1S5 203 25
t. i "''") 1~24|4 342115
N
~
T
Fig. 1. The projection of the left visual field upon the right optic tectum in frog RON 2. The upper diagram represents the outline of the optic tectum seen from above. Each number on the tectum represents an electrode position at which an optimal response was produced when the stimulus was at the position indicated by the same number in the visual field. The perimetric chart extends radially for 100° from the centre of the visual field which is the 'fixation point' of the frog's left eye. N, S, T, I represent the nasal, superior, temporal and inferior poles of the left visual field. Similar conventions are used in the succeeding figures.
gave no responses f r o m the c o n t r a l a t e r a l t e c t u m ; in 6 o f these less than 50 d a y s h a d elapsed b e t w e e n the o p e r a t i o n on the optic nerve a n d the r e c o r d i n g e x p e r i m e n t , a n d in the 7th a n i m a l the i n t e r v a l was 96 days. A f u r t h e r 2 a n i m a l s gave unclassifiable results in t h a t the r e g e n e r a t e d r e t i n o t e c t a l p r o j e c t i o n arose f r o m diffuse areas o f the visual field a n d no o b v i o u s o r d e r c o u l d be f o u n d in the p r o j e c t i o n . T h e r e m a i n i n g 19 a n i m a l s c o u l d be classified a c c o r d i n g to the degree o f n o r m a l i t y o f the regenerated retinotectal projection. T h e t y p e o f p r o j e c t i o n p a t t e r n described b y G a z e a n d J a c o b s o n 7 as p a t t e r n 1 was seen in 2 animals (Fig. 1, R O N 2). The c h a r a c t e r i s t i c f e a t u r e o f this p a t t e r n o f r e g e n e r a t i o n is t h a t a relatively small r e g i o n o f the retina projects to a c o n s i d e r a b l y larger p a r t o f the d o r s a l tectal surface t h a n is the case in a n o r m a l animal. T h e rem a i n d e r o f the r e t i n a does n o t a p p e a r to h a v e c o n n e c t e d with the t e c t u m . W i t h i n t h e r e g i o n o f r e t i n a t h a t has re-established c o n n e c t i o n with the t e c t u m there is no o b v i o u s o r d e r in the p r o j e c t i o n o f retinal p o i n t s t o the tectum. As in the earlier e x p e r i m e n t s o f G a z e a n d J a c o b s o n 7, the a n i m a l s showing p a t t e r n 1 were r e c o r d e d s o o n after the Brain Research, 21 (1970) 183-195
187
FROG OPTIC NERVE REGENERATION
S
I
Fig. 2. The projection of the left visual field upon the right optic tectum in frog R O N 12
optic nerve lesion. Table II shows that these animals occur early in the experimental time-series. A second type of incomplete restoration of the retinotectal projection, in which the projection was appropriately organized across one tectal axis but not across the other, was described by Gaze and Jacobson 7 as pattern 2. A projection similar to this in type was seen in 2 animals in the present series and Fig. 2 (RON 12) illustrates one of these. In pattern 2 the greater part of the tectal projection arises, as in pattern 1, from only a small region of the retina, but in addition the projection to the rostral tectum from the nasosuperior quadrant of the field (temporo-inferior retina) shows evidence of organization along the mediolateral axis of the tectum but not along its rostrocaudal axis. The projection shown in Fig. 2 is more organized than the pattern 2 projection illustrated by Gaze and Jacobson 7 (Fig. 5); in the present case not only is the projection to the most rostral tectum organized in the mediolateral axis of the tectum, but similarly even within the thin band of retina projecting to the greater part of the tectum there is appropriate organization across the mediolateral tectal axis (dorsoventral axis of field), although organization in the rostrocaudal tectal axis (nasotemporal field axis) is absent. A regenerated retinotectal projection showing a pattern intermediate between pattern 2 and the normal is shown in Fig. 3 (RON 25). In this animal there is still a portion of the retina, corresponding to the superior part of the visual field, which Brain Research, 21 (1970) 183-195
188
. -
R. M. GAZE AND M. J, KEATING
lITTI°TuM7 16
15
12 I
7
14
13
II
10
6
5
T
9
N
T I
Fig. 3. The projection of the right visual field upon the left optic tectum in frog RON 25. The unringed numbers on the perimetric chart represent a series of stimulus positions corresponding to the normal projection of the right visual field upon the left tectum. The ringed numbers represent additionall localized but anomalous stimulus positions projecting to the left tectum.
projects rather diffusely to the greater part of the tectum; but superimposed on this diffuse projection is an approximation to a normal retinotectal projection, organized appropriately in both tectal axes. In 8 animals there was, following regeneration of the optic nerve, a restoration of the normal retinotectal projection (Fig. 4, R O N 5). This type of regenerated projection was termed pattern 3 by Gaze and Jacobson 7. Pattern 4, as described by these authors, consisted of an essentially normal retinotectal projection with, in addition, arising from the nasosuperior periphery of the visual field, a retinotopically organized but anomalous projection to the rostral part of the contralateral rectum. In the present experiments this pattern was found in 5 animals, one of which is illustrated in Fig. 5 ( R O N 20). It m a y be seen that electrode positions in the rostral half of the contralateral tectum received input f r o m two positions in the visual field of one eye; one of these positions is that which normally projects to the contralateral tectum while the other, arising f r o m the nasosuperior periphery of the field (ringed numbers), comprises, with its fellows, a retinotopically Brain Research, 21 (1970) 183-195
189
FROG OPTIC NERVE REGENERATION
,,41--
,~0
49
48
46
45
44
43
42
41
47 40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
I0
9
8
7
6
~
17 9
S
44
"~
3~, )5 ~6 i ~
\,
I
__
I
'~,.
'",h 48 39 31
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';, '~
3
1 I~
13
0
Fig. 4. The projection of the right visual field upon the left optic tectum in frog R O N 5.
organized projection which is reversed along the nasotemporal field axis when compared with the normal contralateral projection. Thus in the normal projection (which is approximated by pattern 3 ; Fig. 4) as the electrode positions move caudally on the tectum the corresponding stimulus positions in the visual field move more temporally; but in the anomalous projection as the electrode positions move caudally, the stimulus positions in the field move nasally. Pattern 4 was only found after nerve section, never after nerve crush. All 5 animals showing pattern 4 were mapped a considerable time after the nerve lesion was made; Table [I shows that these patterns all occurred late in the experimental time-series. In all 5 animals in which pattern 4 was found, an anomalous visual projection was also found from the eye to the ipsilateral tectum (Fig. 6, R O N 20). In these animals there was reconstitution of a normal ipsilateral projection from the nasosuperior visual field to the rostral part of the ipsilateral tectum; there was also, however, an additional anomalous but retinotopically organized projection from the whole visual field to the ipsilateral tectum. In its organization this anomalous ipsilateral projection resembles the normal contralateral projection, with nasal field projecting to rostral rectum and temporal field to caudal tectum. Brain Research, 21 (1970) 183-195
190
R. M. GAZE AND M. J. KEATING
RIGHT
TECTUM
17
18
19
20
12
13
14
15
16
8
9
10
11
4
5
6
7
N
"1"
! I
Fig. 5. The projection of the left visual field upon the right optic tectum in frog RON 20. The unringed numbers on the perimetric chart represent the normal contralateral field projection. The ringed numbers constitute an additional series of stimulus positions to the right tectum and forming an additional but anomalous retinotopic projection.
In 1 o f the 5 animals with pattern 4 regeneration ( R O N 28) the pattern o f regeneration to the two tecta was dissimilar. The projection f r o m the left eye to the right tectum consisted o f a mirror-image ipsilateral projection together with a contralateral projection o f pattern 1 type. In this animal the projection f r o m the left eye to the left tectum was straightforwardly pattern 4; that is, there was a full but a n o m a lous contralateral projection to this t e c t u m together with a restored n o r m a l ipsilateral projection. DISCUSSION F o r reasons discussed by M a t u r a n a e t al. 13 and Gaze and J a c o b s o n 7 we believe that normally when recording f r o m the superficial layers o f the contralateral optic tectum we are recording activity f r o m the terminal arborizations o f optic nerve fibres. The m a p p i n g technique used thus indicates the distribution o f optic fibre terminals over the surface o f the tectum at various periods during regeneration o f the optic nerve. Brain Research, 21 (1970) 183-195
FROG OPTIC NERVE REGENERATION
191
s
7
16
I
Fig. 6. The projection of the left visual field upon the left optic tectum in frog RON 20. In this case the ringed numbers on the perimetric chart represent the normal field projection to the ipsilateral tectum whereas the unringed numbers constitute another series of stimulus positions also projecting to the left tectum and forming an additional but anomalous retinotopic projection.
The present results confirm and extend the observations of Gaze and Jacobson v. Negative results preponderate a m o n g those experiments with the shortest o p e r a t i o n recording intervals. The mean interval for those animals which gave no results was 49 days. Presumably the terminal mapping experiment t o o k place before the regenerating optic fibres had reached the rectum. Pattern 1 regeneration tends to be found after short operation-recording intervals. Since the original description of this pattern of early regeneration 7 evidence of similar 'spreading' of developing or regenerating optic nerve fibres over the tectum has been sought unsuccessfully in other species. The failure to demonstrate this spreading at the tectal level by means of classical histological methods in goldfish 2 or embryonic chicks 4 is not too surprising since the terminal fibres may be very small and dispersed and may hardly be traced with ordinary silver stains. Electrophysiological studies in fish 1°,zl have, however, also failed to demonstrate any spreading of optic nerve fibres. The two animals showing pattern 1 regeneration in this series confirm, however, that in the frog, during early stages of regeneration of the optic nerve, fibres f r o m Brain Research, 21 (1970) 183-195
192
It, M. G A Z E A N D M. J. K E A T I N G
small areas of the retina may spread over considerably greater areas of tectum than those to which they normally project. The work of Cronly-Dillon 3 on the regeneration of retinotectal connections in newts suggests that a comparable phenomenon may occur in urodeles. This author found that the multi-unit receptive field size of a given tectal locus was larger than normal during the earlier stages of regeneration; and this was particularly so in the case of projections from the peripheral regions of the retina. On the other hand the fact that in urodeles the entire retina degenerates and then regenerates after optic nerve lesions complicates interpretation of the results in these animals and, as Gaze and Watson 9 have shown, the regenerated central retina in newts is older than the regenerated peripheral retina. Gaze and Jacobson 7 suggested that the various patterns of regeneration, in numerical order, represented sequential stages in the restoration of the retinotectal projection. Our results would support this idea on two counts: firstly the time distribution of the various patterns in the present experiments suggests that pattern 1 is an early, and pattern 3 or 4 a late, manifestation of the regeneration process; and secondly the 2 animals classed as pattern 2 in the present series in fact represent intermediate stages between patterns 2 and 3. If the pattern being described represents successive stages in the process of regeneration, then the progression from pattern 1 to pattern 2 and thence to normal (pattern 3) indicates that, following the initial diffuse projection from small retinal areas, as more fibres regenerate to the tectum, the organization of these fibres occurs in stages. The first semblance of organization occurs at the rostral pole of the tectum and the organization then spreads over the rest of the tectum. This rostrocaudal tectal sequence of organization parallels the rostrocaudal sequence of stratification and acetylcholinesterase appearance during development and in both cases the rostrocaudal direction of spread is presumably related to the fact that the optic axons grow in from the rostral end of the tectum. It would be interesting in this context to observe the result of growing an optic nerve into the tectum from an abnormal direction. It seems likely that during the process of regeneration of the optic fibres order arises out of the initially diffuse early patterns due to a process of competition for appropriate tectal positions by the regenerating fibres. Thus the first fibres to regenerate spread diffusely over the tectum since there are no other fibres with which to compete. As larger numbers of fibres arrive competition increases and aberrant connections will be displaced as fibres more appropriate to the tectal locus arrive. Large numbers of regenerating fibres will arrive first at the rostral pole of the tectum and thus semblances of organization appear here first. Direct evidence for the competitive re-innervation of tectal loci is provided by Gaze and Sharma 8 and a similar process of competition is suggested by Cronly-Dillon a. It is not immediately apparent, however, why the regenerating fibres become organized along the mediolateral axis of the tectum (dorsoventral field axis) before they do along the rostrocaudal tectal axis (nasotemporal field axis). It is conceivable that this axial difference reflects the mode of ingrowth of regenerating fibres. If Brain Research, 21 (1970) 183-195
FROG OPTIC NERVE REGENERATION
193
OPTIC
Fig. 7. See text.
we conceive of the main mass of fibres advancing rostrocaudally over a wide front and these fibres have branched, then along any mediolateral strip of rostral tectum there will be a chance of fibre competition occurring before it can occur in the rostrocaudal axis. In this case the axial differences noted would reflect the temporal sequence and direction of innervation. Many fibres arriving more or less simultaneously at level AB (mediolateral axis, Fig. 7) will be able to start the sorting process; whereas sorting along the rostrocaudal axis (Fig. 7 BC) cannot yet start, since the main fibre mass has not yet reached C. Fibre competition and tectal axial differences are further considered by Gaze and Sharma s. Eight animals showed a normal retinotectal projection following regeneration of the optic nerve. In these cases the operation-recording interval varied from 40 to 396 days (Tables I and II). Five animals gave the intriguing pattern 4. This pattern consists, in a way, of a 'supernormal' projection, in that in addition to the restored normal retinal projection to the contralateral rectum there is a retinotopically organized projection from the nasosuperior periphery o f the field to the rostral tectum (Fig. 5). This anomalous contralateral projection is in fact an ipsilateral projection which has appeared in the 'wrong' tectum 6,v. More will be said about the significance of the anomalous pattern 4 projection in a subsequent paper on the restoration of the ipsilateral visual projection following regeneration of the optic nerve. Here we need say only that the basic abnormality in this type of regeneration pattern is that the regenerating nerve fibres appear to regenerate directly not only to the contralateral tectum but also to the ipsilateral tectum; and they thus produce mirror-image contralateral-type projections in both tecta (Figs. 5 and 6). It was suggested by Gaze and Jacobson v that pattern 4 may arise as a progression from pattern 3 in that the former is a 'super-complete' type of regeneration. However, a consideration of Table II indicates that patterns 3 and 4 are probably not sequential stages of regeneration but are alternative endpoints of the regeneration process, dependent upon the type of operation used to produce the lesion in the optic nerve. Crush lesions, which leave the sheath of the nerve intact, lead to pattern 3; nerve sections, which permit aberrant fibres to enter the diencephalon ipsilaterally, lead Brain Research, 21 (1970) 183-195
194
R. M. GAZE AND M. J. KEATING
t o p a t t e r n 4. T h e ability o f r e t i n a l axons t o f o r m a p p r o p r i a t e c o n n e c t i o n s with either tectum, i f given the o p p o r t u n i t y , was i l l u s t r a t e d b y S p e r r y 16 w h o d e l i b e r a t e l y crossu n i t e d o p t i c nerves to the i p s i l a t e r a l optic t r a c t . SUMMARY
T h e c o n t r a l a t e r a l r e t i n o t e c t a l p r o j e c t i o n was m a p p e d in a series o f frogs at v a r i o u s intervals after i n t e r r u p t i o n o f the optic nerve. F u r t h e r evidence o f a n early diffuse stage o f r e g e n e r a t i o n has been o b t a i n e d . F o l l o w i n g this diffuse stage there a p p e a r s to o c c u r an o r g a n i z a t i o n o f the tectal c o n n e c t i o n s o f o p t i c fibres initially a l o n g the m e d i o l a t e r a l axis o f the t e c t u m a n d subsequently a l o n g the r o s t r o c a u d a l tectal axis. Some m o r e i n t e r m e d i a t e p a t t e r n s o f r e g e n e r a t i o n h a v e been d e s c r i b e d s u p p o r t i n g the view t h a t these p a t t e r n s are stages in a c o n t i n u o u s sequence. T h e occurrence o f p a t t e r n 4 r e g e n e r a t i o n to t h e c o n t r a l a t e r a l t e c t u m has been s h o w n to be a s s o c i a t e d i n v a r i a b l y with a n o m a l o u s r e g e n e r a t i o n to the i p s i l a t e r a l tectum. It is suggested t h a t p a t t e r n 3 a n d p a t t e r n 4 are a l t e r n a t i v e e n d - p o i n t s o f the r e g e n e r a t i o n sequence, d e p e n d e n t u p o n the t y p e o f o p e r a t i v e p r o c e d u r e on the optic nerve. ACKNOWLEDGEMENT
W e are grateful t o M i s s E. F o r r e s t f o r h e r excellent h i s t o l o g i c a l assistance.
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Astronotus ocellatus, Developm. Biol., 7 (1963) 234-243. 2 ATTARDI, D. G., AND SPERRY, R. W., Preferential selection of central pathways by regenerating
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