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Specification of regenerating retinal ganglion cells in the adult newt,triturus cristatus
Brain Research, 68 (1974) 319-329 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
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S P E C I F I C A T I O N OF R E G E N E R A T I N G R E T I N A L G A N G L I O N CELLS I N THE ADULT NEWT, TRITURUS CRISTATUS
R. L. LEVINE* AND J. R. CRONLY-DILLON
Department of Ophthalmic Optics, University of Manchester Institute of Science and Technology, Manchester 1 (Great Britain) (Accepted September 18th, 1973)
SUMMARY Experiments were carried out with Triturus cristatus to try to locate those structures within the eye that determine the specification of the retina during regeneration in the adult animal. In the present study lens and retina were removed and the anterior part of the eye surgically rotated 180 ° before reattaching to the posterior unrotated portion of the eye. In one group of animals, where the incision was made some distance behind the limbus (post-limbal animals), the rotated anterior part of the eye included the cornea, iris, ciliary margin, and a peripheral band of pigment epithelium. In another set of experiments, the incision was made along the limbus, destroying the ciliary margin. Here the anterior part now contained only cornea, iris, and little if any pigment epithelium (limbal animals). After allowing enough time for the lens, retina and optic nerve to regenerate, the visuotectai projection was mapped electrophysiologically with Woods metal microelectrodes. Two types of visuotectal projections were found. In post-limbal animals, almost half the group showed completely rotated visuotectal projections. The remainder gave maps in which the tectal representation of the peripheral area of the visual field was rotated 180 ° but the representation of the central part of the visual field projected to the tectum in a normal fashion. In limbal animals the two types of visuotectal maps were again seen, but here only a small percentage of animals showed completely rotated visuotectal projections. The majority of animals in this group gave the second type of map where an unrotated representation of the central region of the visual field was combined with a rotated representation of the visual field periphery. These * Present address: Department of Physiology and Biophysics, University of Miami, School of Medicine, P.O. Box 875, Biscayne Annex, Miami, Fla. 33152, U.S.A.
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findings suggest that both the iris, and the pigment epithelium in the posterior part of the eye each have the capacity to specify regenerating retina. However, under certain conditions the anterior portion of the eye may be dominant, and specify the entire new retina. None of the animals in either group gave completely unrotated projections. This suggests that the posterior part of the eye is not able to override the specifying influence of the anterior portion.
INTRODUCTION
During amphibian embryogenesis the developing retina becomes polarized, and this event predisposes the future axons of its constituent retinal ganglion ceils to terminate in a topographically orderly and predictable fashion, when they reach the visual centres of the brain 11. When the retina has been polarized in this way its retinal ganglion cells are said to have been specified with respect to the relative positions each occupies in the retina. Retinal polarization and the specification of retinal ganglion cells has also been studied in adult urodeles. The latter are unique among the vertebrates, in being able to regenerate a new retina from the pigment epithelium and the pars ciliaris retinae 7, la-15 following surgical removal of the original retina. Using electrophysiological methods, Burgen and Grafstein 1 and Cronly-Dillon 2 demonstrated in urodeles that the distribution of the recordable portions of optic axons (presumably the terminal arbors3,4,16), in the optic tectum, is essentially normal following retinal regeneration (in the case of Burgen and Grafstein's work, this was only true if the disposition of various parts of the eye had not been altered after retinal removal - - see below). This pattern of distribution of optic afferents on the tectum is referred to as the visuotectal projection (VTP). The presence of a normal visuotectal projection after retinal regeneration suggests that the original, preoperative polarity of the retina has been restored. Following retinal removal, the source of positional information for the specification of regenerating retinal ganglion ceils might either reside within the eye itself 9 or might be present in the extraocular tissuesS, ~2. One way of distinguishing between these two possibilities, is to alter the position of the eye, with reference to the body axes, before regeneration of the retina has occurred. While this particular experiment has not yet been done, the work of Grafstein and Burgen a,6 may serve as an adequate substitute. These workers divided the eye of the adult newt, Notophthalmus viridescens, into an anterior and a posterior part, by making an incision a short distance behind the corneo-scleral junction and running along the entire circumference of the eye. The retina was removed from both parts of the eye and the anterior part of the eye was then rotated 180 ° in the plane of the incision and replaced onto the posterior part. Allowing 4-7 months for retinal regeneration the visuotectal projection was then mapped in 12 of these animals. The results of these experiments indicated that some form of competitive interaction had occurred between the anterior and posterior parts of the eye, in specifying
REGENERATING RETINAL GANGLION CELLS IN THE NEWT
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the ganglion cells of the regenerating retina. In some cases they found that the anterior part of the eye appeared to have specified the entire retina (completely rotated VTPs) while in others, the posterior part of the eye appeared to have played the dominant role (unrotated VTPs). Finally, 2 of the 12 animals which they examined showed some evidence of an interaction between the two parts of the eye. In these animals the map now included a representation of the central part of the field in an abnormal position on the tectum. Unfortunately, the visuotectal maps were not sufficiently well organized topographically to determine the orientation of each visual field representation. The conclusion we have drawn from the work of these authors is that the anterior part of the eye, in the adult newt, is capable of directing the specification of the retinal ganglion cells of an entire regenerated retina. It is also evident from their work that some form of interaction may occur between the anterior and posterior parts of the eye during the regenerative process. The experiments in the present paper were undertaken with the following objectives in mind. (a) First, we intended to repeat the experiments of Burgen and Grafstein, in Triturus cristatus, in the hope of confirming and perhaps extending their results. (b) Secondly, it was hoped that we would be able to localize, more effectively, the source of positional information in the anterior part of the eye of the adult newt. MATERIALS AND METHODS
(1) Experimental animals The newts used in these experiments were adult T. cristatus, obtained from a commercial dealer. The animals were kept in ordinary fish tanks which were filled with tap water and kept at room temperature. A constant supply of Tubifex worms was kept in the tanks as food for the experimental animals.
(2) Surgical procedures (a) Eye operations. Prior to surgery, anaesthesia was effected by immersion in 0.4 ~ MS 222 (Sandoz) for 4-10 min. The animal was then placed in a plasticine holder and immersed in Holtfreter's solution. All ocular surgery was performed with the animal submerged. In one group of animals an incision was made entirely around the globe, at some distance behind the limbus corneae (corneo-scleral junction). These operations were similar to those performed by Burgen and Grafstein 1. In a second group of animals, the incision was made along the limbus. All incisions were made using a capsuiotomy scissors. After the incision had been made, the retina and lens were removed from the dissected eye. Removal of the retina was facilitated by the fact that it tended to float free of the retinal pigment epithelium (PE), when the eye had been opened. Removal was completed by tearing the remaining retinal attachments at the optic disc and the ciliary margin. Occasionally, small pieces of retina would remain attached at these two sites after the removal procedure. Such remnants could
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R. L. LEVINE A N D J. R. C R O N L Y - D I L L O N
Post-limbal Incision
Lens and Retina Removed
Anterior Part Rotated and Reattachedto Posterior Part
Limbol Incision
Fig. 1. Schematic diagram showing operations described in text. Note the ventral notch in the pupil and the dorsoventrally asymmetric band of pigment along the limbus. Both of these characteristics were used to determine the orientation of the anterior part of the eye, during surgery and postoperatively.
be easily seen and were removed with watchmaker's forceps or by suction through a micropipette. The lens was also removed. After lens and retina were removed, the anterior part of the eye was rotated 180 °, in the plane of the incision, and returned to the posterior part of the eye. These operations are shown diagrammatically in Fig. 1. A series of control operations was also done, in which the anterior part of the eye was not rotated. The terms limbal and post-limbal will be used to describe respectively, (1) operations where an incision has been made along the limbus and (2) operations where the incision was made some distance behind the limbus. Correspondingly, operations in which the anterior part of the eye has been rotated will be referred to as either limbal or post-limbal rotations, depending on where the incision to the eye was made. After the operation, each animal was allowed to recover, for 3 days, in a moist container kept at 10 °C. Animals were then transferred to their home tanks at room temperature. (b) Preparation of anirnalsfor recording. Before recording, animals were immobilized with 0.18-0.25 ml 4 ~ Flaxedil (gallamine), 1 h before surgery. Immediately prior to the operation, they were anaesthetized with 0.4 ~ MS 222, for 3 min. The animal's skull was exposed through a longitudinal incision in the overlying skin. A rectangular bone flap was made, over the tectum, using a dental drill and the flap was reflected, exposing the brain and meninges. A drop of mineral oil was placed on the exposed tissues. The dura and subdural membranes were torn and reflected, exposing the tectum for recording.
REGENERATING RETINAL GANGLION CELLS IN THE NEWT
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Four animals, which had been subjected to post-limbal rotations, were recorded from between 3 and 4 months postoperatively. All other experimental animals were recorded from between 7 and 12 months after their operations.
(3) Recording procedures The microelectrodes used in this study were Woods metal filled micropipettes, Gestland et al. 5, with tip diameters of 2-5 btm. The electrode tips were plated with a ball of platinum black immediately prior to recording. The earth electrode was a stainless steel hypodermic needle which was inserted in the tail of the animal. The input stage of the amplification system was a Transidyne MPA-6 differential preamplifier which was used single ended, (input impedance of this device was 1011 f2). After suitable amplification, the signal was processed through a noise blanker which only passed pulses whose amplitude exceeded a preset level. From here the signal was relayed to an oscilloscope and to a loudspeaker.
(4) Mapping procedures After exposure of the optic tecta, the animal was placed on a cork platform and its eye was centred on the centre of a projection perimeter. The microelectrode was moved in 125 # m steps, across the surface of the tectum and the visual field position giving maximum responses at each tectal position, was noted. RESULTS
In normal animals, multiunit response field diameters ranged from 5-10 ° at the centre of the visual field to 15-20 ° in the periphery. In operated animals the response fields were consistently larger, usually ranging from 20 ° to 40 ° in diameter. In addition, in operated animals, there was no clearcut gradation of response field sizes, as one moved from the centre to the periphery of the visual field. The differences between response field sizes in normal and operated animals is presumably the result of abnormally extensive branching of the terminal arbors of the optic nerve fibres after retinal regenerationL However, because single unit recording was not done in the present experiments, one cannot say with certainty whether or not the enlarged multiunit response fields in operated animals were due, at least in part, to an increase in the size of individual single unit receptive fields. The normal VTP, of T. cristatus, is shown in Fig. 2. The visual field rows run ventrodorsally from their numbered extremities, which corresponds to a lateromedial progression of the points in the tectal rows. The most nasal portion of the visual field (row 1) is projected to the most anterior portion of the tectum, while the most temporal portion (row 8) is projected posteriorly. Only the dorsal part of the visual field is projected to the exposed portion of the tectum, the centre of the field being represented near its posterolateral pole. Studies in other species 1° indicate that the remainder of the visual field projects to the lateroventral, unexposed portion of the rectum. In animals that had undergone limbal or post-limbal operations, without rota-
324
R.
L.
LEVINE
AND
J.
R.
CRONLY-DILLON
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D
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v
ns~m ~
Left Visual Field
~ Right Tectum
Fig. 2. Visuotectal projection (VTP) recorded from a normal, unoperated animal. The numbered rows of tectal loci (open circles) receive their input from correspondingly numbered rows of loci in the visual field.
tion o f the anterior p a r t o f the eye, the p o l a r i t y o f the regenerated VTP was always n o r m a l . However, if the a n t e r i o r p a r t o f the eye h a d been rotated, the VTP was always a b n o r m a l . Since animals with r o t a t i o n o f the a n t e r i o r p a r t o f the eye gave similar results regardless o f whether a limbal or p o s t - l i m b a l incision h a d been used, these results will be described initially w i t h o u t reference to the type o f incision. T w o m a j o r classes o f results were f o u n d in animals with the a n t e r i o r p a r t o f the eye rotated. A n example o f the first class o f results is shown in Fig. 3. N o t e that the field rows run d o r s o v e n t r a l l y in the visual field. Also, the most nasal field row D
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Fig. 3. Visuotectal projection of an animal in which a post-limbal rotation was performed 9 months previously. Note that the projection is completely inverted. Vertically divided circles represent tectal loci which did not respond to a visual stimulus.
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REGENERATING RETINAL GANGLION CELLS IN THE NEWT
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Fig. 4. Visuotectal projection of an animal in which a limbal rotation was performed 10 months previously. Note that there is a dorsal portion of the visual field which projects to the tectum with normal polarity (open circles). The ventral and lateral portions of the visual field project to the tectum with rotated polarity (filled circles). Rows 4 and 8, on the tectal surface, each receive input from two field rows in the visual field. Points on the tectum, which receive input from two loci in the visual field, are represented by horizontally divided circles. Some field rows in the rotated portion of the map are separated, on the surface of the tectum and in the visual field, into two segments, by the unrotated portion of the map (see rows 2 and 3). In these cases, the most medial segment on the tectal surface is denoted by an asterisk.
(row 8) projects to the p o s t e r i o r p a r t o f the tectum while the t e m p o r a l pole o f the visual field (row 1) is referred to the a n t e r i o r p a r t o f the tectum. These c o n d i t i o n s are the inverse o f those seen in the n o r m a l a n i m a l (see Fig. 2). Because o f this inversion, there is n o w a sizeable p o r t i o n o f the ventral visual field which projects to the d o r s a l surface o f the tectum. I n some a n i m a l s the a n t e r i o r p a r t o f the eye was r o t a t e d slightly m o r e or less t h a n 180 ° . I n these cases the visual field r e p r e s e n t a t i o n deviates f r o m a full 180 ° r o t a t i o n b y an a m o u n t c o m m e n s u r a t e with the e r r o r in the surgical r o t a t i o n . T h e field rows in m a p s which show c o m p l e t e inversion o f the V T P are generally well o r d e r e d t o p o g r a p h i c a l l y relative to each o t h e r a n d show little o v e r l a p with a d j a c e n t rows. T h e second class o f results f o u n d also in cases where the a n t e r i o r p a r t o f the eye h a d been rotated, consisted o f m a p s in which the visual field p r o j e c t i o n to the t e c t u m has divided into two t o p o g r a p h i c a l l y distinguishable parts. A n e x a m p l e o f this is seen in Fig. 4. The peripheral portions o f ventral, nasal, a n d t e m p o r a l areas o f the visual field in this a n i m a l are seen to project to the tectum with r o t a t e d polarity. I n the d o r s a l region o f the visual field, however, there is an area which projects with n o r m a l p o l a r i t y to the tectum. This a r e a fits into the space between the nasal a n d t e m p o r a l limbs o f the r o t a t e d p a r t o f the VTP. I t is n o t e w o r t h y t h a t we f o u n d no cases in the visual field where the two parts o f the m a p were superimposed. Indeed, the two m a p s a p p e a r to be m u t u a l l y exclusive in the visual field. H o w e v e r , one o c c a s i o n a l l y finds a slight o v e r l a p o f the field p r o j e c t i o n s on the tectal surface. This
326
R. L. LEVINE AND J. R. CRONLY-DILLON Caudal D
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Fig. 5. Visuotectal projection o f an animal in which a post-limbal rotation was performed 7 m o n t h s previously. Note that there is a central, unrotated portion and a peripheral, rotated portion to the visual field map. Symbols as in Fig. 4.
can be seen, in Fig. 4, for those tectal portions represented by the horizontally divided circles. Each divided circle on the tectal surface represents a recording point which received its input from two areas, in the visual field. One of these areas was always part of the rotated projection and the other was part of the normal map. This is most clearly seen in visual field rows 8 and 8', which are widely separated in the visual field but project to the same row of recording points on the tectum. Another example of a two-part map is shown in Fig. 5. In this map the normal portion is centrally located and is almost completely surrounded by a peripheral band of the visual field which projects to the tectum with rotated polarity. This type of result was seen to occur in animals that had been subjected to post-limbal rotations. As in the previous example, the two parts of the map are mutually exclusive in the visual field and show only slight overlap on the tectal surface. In both types of two part maps, i.e., those which display a dorsally displaced normal portion as distinct from those with a normal portion that is located in the central part of the visual field, the field rows of the rotated map tend to be arranged topographically in an orderly fashion, except in the ventral part of the field where some overlap of rows may occur. The field rows of the normally projecting part of the map are often less regularly arranged than those of the rotated map, and even when they are regularly disposed with respect to one another, they are often distorted at the boundary which divides them from the rotated portion of the map (see Fig. 4). In no case did we observe a complete map with normal polarity and ordering, in animals that had undergone limbal or post-limbal operations where the anterior part of the eye was rotated. Recordings were made from 16 animals that had undergone post-limbal rotations. Of these, 8 showed complete rotation of the VTP, while another 8 gave two-part maps. Of the latter, 3 had dorsally displaced, portions of the visual field that projected
327
REGENERATING RETINAL GANGLION CELLS IN THE NEWT TABLE I
Operations
Post-limbal Limbal
Total number of animals
16 13
Types of maps recorded Complete rotation
8 3
Two part maps Dorsal normal portion
Central normal portion
Other
3 7
4 --
l* 3
* T h e m a p p i n g p r o c e d u r e was n o t c o m p l e t e d in this a n i m a l a n d the disposition o f the n o r m a l p o r t i o n o f t h e m a p c a n n o t be ascertained f r o m the d a t a which were recorded.
normally, while in 4 animals the normal projection was centrally located. In 1 animal the map we obtained was not complete enough to enable us to determine the disposition of the normal field. Of 13 animals with limbal rotations, only 3 showed completely rotated VTPs. The remaining 10 gave two-part maps of which 8 had dorsally displaced, normal portions. In 3 of these 8 animals, the putative normal portion of the visual field contained systematic abnormalities. Of the 2 remaining animals, 1 gave a single normally oriented row which was displaced ventrally in the visual field, the other animal had a large dorsally displaced normal field which extended ventrally to bisect the rotated portion of the field. These results are summarised in Table I. DISCUSSION
The purpose of this study was to try to localize the structure, or structures, responsible for specifying regenerating retinal ganglion cells in the eye of the adult newt. In all cases where the anterior part of the eye was rotated through 180 °, some or all of the regenerated VTP was found to be rotated. This result was obtained regardless of whether the incision freeing the anterior part of the eye had been made at, or behind the limbus. Control animals, where the anterior part of the eye had been replaced in a normal orientation, showed VTPs with normal polarity. This indicates that inversions of the map seen when the anterior part of the eye was rotated were not attributable to operative trauma. Thus, in animals that have undergone limbal or post-limbal rotations, the anterior part of the eye always appears to play a role in the specification of the regenerated retinal ganglion cells. Though many animals where the anterior part of the eye was rotated showed normally oriented portions of the VTP, none of them showed a completely unrotated map. In animals where we found two part maps, the normally projecting part was usually smaller than the rotated part and occasionally projected to only a portion of a single tectal row. These findings indicate that while the posterior part of the eye or the extraocular tissues may specify retinal cells, their 'specifying' effect on the entire regenerating retina is less predictable and not as strong as that of the anterior part. Since the unrotated source(s) of positional information exhibit such erratic
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R. L. LEVINE AND J. R. CRONLY-DILLON
behaviour in determining the polarity of the regenerating retina, one might expect such sources to be localized in a highly labile structure. The retinal PE is an obvious candidate for this role, since it is responsible for the regeneration of part of the retina and undergoes proliferative and metaplastic changes during this process7,1~-15. In addition, the extent of the retinal PE sheet initially remaining in the posterior part of the eye, varies from animal to animal, as a result of operative trauma 15. Thus the retinal PE, in the posterior part of the eye, is both labile and variable in extent and it is plausible to suggest that it may be responsible for specifying those cells in the retina that give rise to the normal portions of the two part maps. To test this hypothesis specifically would require one to repeat the present operations combined with removal of the PE from the posterior part of the eye. Such experiments are in progress and will be reported in a later paper. The simplest explanation for the two types of maps found in this study, i.e., two part maps and completely rotated maps, is that they represent varying amounts of growth of retinal tissue derived from the anterior, rotated part of the eye onto the posterior unrotated part. The completely rotated maps would then represent cases where the entire regenerated retina was derived from the anterior, rotated portion of the eye. If this explanation is the correct one, evidence of varying amounts of invasion of the posterior part of the eye by tissue derived from the anterior part should be available from histological studies. Indeed, histological data from the present experiments 15, indicate that in eyes that have been subjected to post-limbal operations there is a good deal of growth of the unpigmented cells of the anterior part of the regenerate onto the posterior part of the eye. The unpigmented lamina continues to spread until it is arrested by contact with regenerating retina derived from the posterior PE. Because regenerating retina in the posterior part may be at various stages of development when contact between the two occurs, the extent of spreading of the unpigmented cells varies from one animal to the next. However, in no case was the tissue derived from the anterior part seen to cover the entire posterior surface of the eye. Thus, although these observations may account in part for the observed results, they do not explain the occurrence of totally rotated maps which were found in 50 ~ of the animals with post-limbal rotations. Histological evidence suggests that in limbal eyes the anterior part of the eye is no longer the dominant source of new retinal ganglion cells 15. In addition a limbal incision effectively removes any positional cues, present in the retinal PE, from the anterior part of the eye. The occurrence of rotated visuotectal projections from such eyes therefore implies that some of the positional information imparted to the regenerating retina must derive from the regenerative tissues of the iris. In those animals, subjected to limbal rotations, which show completely rotated VTPs, the anterior part of the eye has specified an entire set of retinal ganglion cells, the majority of which presumably are derived from the unrotated retinal PE l~. Thus, the influence of the anterior part of the eye, on the specification of regenerating retinal ganglion cells, would appear to be able to extend into tissue not derived from the anterior part of the eye.
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329
Thus, there a p p e a r to be two i n d e p e n d e n t sources o f p o s i t i o n a l i n f o r m a t i o n in the eye o f the a d u l t newt. One source t h a t is l o c a t e d in the a n t e r i o r p a r t o f the eye, p r e s u m a b l y in the iris. P o s i t i o n a l i n f o r m a t i o n derived f r o m this region a p p e a r s to be c a p a b l e o f overriding a n y other p o s i t i o n a l i n f o r m a t i o n present in the eye. The second source is l o c a t e d in the p o s t e r i o r p a r t o f the eye, p e r h a p s in the retinal p i g m e n t epithelium. P o s i t i o n a l i n f o r m a t i o n derived f r o m the latter seems less stable t h a n t h a t derived f r o m the a n t e r i o r p a r t o f the eye a n d m a y indeed be o v e r r i d d e n b y i n f o r m a t i o n issuing f r o m t h a t part. ACKNOWLEDGEMENTS This research was s u b m i t t e d to the D e p a r t m e n t o f Biology b y R. L. Levine at Yale U n i v e r s i t y in p a r t i a l fulfillment o f the requirements for a Ph.D. degree. One o f us (R. L. L.) was s u p p o r t e d d u r i n g this research b y U S P H S Traineeship 5T01 HD-00032-09.
REFERENCES 1 BURGEN,A. S. V., AND GRAFSTEIN,B., Retinotectal connections after retinal regeneration, Nature (Lond.), 196 (1962) 898-899. 2 CRONLY-DILLON,J. R., Pattern of retinotectal connections after retinal regeneration, J. Neurophysiol., 31 0968) 410-418. 3 GAZE, R. M., The Formation of Nerve Connections, Academic Press, New York, 1970. 4 GEORGE,S. A., Studies on Optic Nerve Terminal Arborizations in the Frog's Tectum, Ph.D. Thesis, Johns Hopkins Univ., Baltimore, Md., 1970. 5 GESTLAND,R. C., HOWLAND,B., LETTVIN,J. Y., ANDPITTS,W. H., Comments on microelectrodes, Proc. Inst. Radio Engrs, 47 (1959) 1856-1862. 6 GRAFSTEIN,B., AND BURGEN,A. S. V., Pattern of optic nerve connections following retinal regeneration. In W. BARGMANNAND J. P. SCHADI~(Eds.), Topics in Basic Neurology, Progress in Brain Research, Vol. 6, Elsevier, Amsterdam, 1964, pp. 126-138. 7 HASEGAWA,M., Restitution of the eye after removal of the retina and lens in the newt, Triturus pyrrhogaster, Embryologia (Nagoya), 4 (1958) 1-32. 8 HUNT, R. K., ANDJACOBSON,M., Development and stability of positional information in Xenopus retinal ganglion cells, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 780-783. 9 HUNT, R. K., AND JACOBSON,M., Specification of positional information in retinal ganglion cells of Xenopus: Assays for analysis of the unspecified state, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 507-511. 10 JACOaSON,M., The representation of the retina on the optic tectum of the frog. Correlation between retinotectal magnification factor and retinal ganglion cell count, Quart. J. exp. Physiol., 47 (1962) 170-178. 11 JACOBSON,M., Retinal ganglion cells: specification of central connections in larval Xenopus laevis, Science, 155 (1967) 1106-1108. 12 JACOBSON,M., Development of neuronal specificity in retinal ganglion cells of Xenopus, Develop. Biol., 17 (1968) 202-218. 13 KEEFE, J. R., An analysis of urodelian retinal regeneration: I. Studies of the cellular source of retinal regeneration in Notophthalmus viridescens utilizing aH-thymidine and colchicine, J. exp. Zool., 184 (1973) 185-206. 14 KEEFE, J. R., An analysis of urodelian retinal regeneration: IV. Studies of the cellular source of retinal regeneration in Triturus cristatus carnifex using aH-thymidine, J. exp. Zool., 184 (1973) 239258. 15 LEVINE,R., Regeneration of the retina in the adult newt, Triturus cristatus, after surgical division of the eye, In preparation. 16 MATURANA,H. R., LETTVIN,J. Y., McCULLOCH, W. S., AND PITTS,W. H., Anatomy and physiology of vision in the frog (Rana pipiens), J. gen. Physiol., Suppl. 43 (1960) 129-175.
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S P E C I F I C A T I O N OF R E G E N E R A T I N G R E T I N A L G A N G L I O N CELLS I N THE ADULT NEWT, TRITURUS CRISTATUS
R. L. LEVINE* AND J. R. CRONLY-DILLON
Department of Ophthalmic Optics, University of Manchester Institute of Science and Technology, Manchester 1 (Great Britain) (Accepted September 18th, 1973)
SUMMARY Experiments were carried out with Triturus cristatus to try to locate those structures within the eye that determine the specification of the retina during regeneration in the adult animal. In the present study lens and retina were removed and the anterior part of the eye surgically rotated 180 ° before reattaching to the posterior unrotated portion of the eye. In one group of animals, where the incision was made some distance behind the limbus (post-limbal animals), the rotated anterior part of the eye included the cornea, iris, ciliary margin, and a peripheral band of pigment epithelium. In another set of experiments, the incision was made along the limbus, destroying the ciliary margin. Here the anterior part now contained only cornea, iris, and little if any pigment epithelium (limbal animals). After allowing enough time for the lens, retina and optic nerve to regenerate, the visuotectai projection was mapped electrophysiologically with Woods metal microelectrodes. Two types of visuotectal projections were found. In post-limbal animals, almost half the group showed completely rotated visuotectal projections. The remainder gave maps in which the tectal representation of the peripheral area of the visual field was rotated 180 ° but the representation of the central part of the visual field projected to the tectum in a normal fashion. In limbal animals the two types of visuotectal maps were again seen, but here only a small percentage of animals showed completely rotated visuotectal projections. The majority of animals in this group gave the second type of map where an unrotated representation of the central region of the visual field was combined with a rotated representation of the visual field periphery. These * Present address: Department of Physiology and Biophysics, University of Miami, School of Medicine, P.O. Box 875, Biscayne Annex, Miami, Fla. 33152, U.S.A.
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findings suggest that both the iris, and the pigment epithelium in the posterior part of the eye each have the capacity to specify regenerating retina. However, under certain conditions the anterior portion of the eye may be dominant, and specify the entire new retina. None of the animals in either group gave completely unrotated projections. This suggests that the posterior part of the eye is not able to override the specifying influence of the anterior portion.
INTRODUCTION
During amphibian embryogenesis the developing retina becomes polarized, and this event predisposes the future axons of its constituent retinal ganglion ceils to terminate in a topographically orderly and predictable fashion, when they reach the visual centres of the brain 11. When the retina has been polarized in this way its retinal ganglion cells are said to have been specified with respect to the relative positions each occupies in the retina. Retinal polarization and the specification of retinal ganglion cells has also been studied in adult urodeles. The latter are unique among the vertebrates, in being able to regenerate a new retina from the pigment epithelium and the pars ciliaris retinae 7, la-15 following surgical removal of the original retina. Using electrophysiological methods, Burgen and Grafstein 1 and Cronly-Dillon 2 demonstrated in urodeles that the distribution of the recordable portions of optic axons (presumably the terminal arbors3,4,16), in the optic tectum, is essentially normal following retinal regeneration (in the case of Burgen and Grafstein's work, this was only true if the disposition of various parts of the eye had not been altered after retinal removal - - see below). This pattern of distribution of optic afferents on the tectum is referred to as the visuotectal projection (VTP). The presence of a normal visuotectal projection after retinal regeneration suggests that the original, preoperative polarity of the retina has been restored. Following retinal removal, the source of positional information for the specification of regenerating retinal ganglion ceils might either reside within the eye itself 9 or might be present in the extraocular tissuesS, ~2. One way of distinguishing between these two possibilities, is to alter the position of the eye, with reference to the body axes, before regeneration of the retina has occurred. While this particular experiment has not yet been done, the work of Grafstein and Burgen a,6 may serve as an adequate substitute. These workers divided the eye of the adult newt, Notophthalmus viridescens, into an anterior and a posterior part, by making an incision a short distance behind the corneo-scleral junction and running along the entire circumference of the eye. The retina was removed from both parts of the eye and the anterior part of the eye was then rotated 180 ° in the plane of the incision and replaced onto the posterior part. Allowing 4-7 months for retinal regeneration the visuotectal projection was then mapped in 12 of these animals. The results of these experiments indicated that some form of competitive interaction had occurred between the anterior and posterior parts of the eye, in specifying
REGENERATING RETINAL GANGLION CELLS IN THE NEWT
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the ganglion cells of the regenerating retina. In some cases they found that the anterior part of the eye appeared to have specified the entire retina (completely rotated VTPs) while in others, the posterior part of the eye appeared to have played the dominant role (unrotated VTPs). Finally, 2 of the 12 animals which they examined showed some evidence of an interaction between the two parts of the eye. In these animals the map now included a representation of the central part of the field in an abnormal position on the tectum. Unfortunately, the visuotectal maps were not sufficiently well organized topographically to determine the orientation of each visual field representation. The conclusion we have drawn from the work of these authors is that the anterior part of the eye, in the adult newt, is capable of directing the specification of the retinal ganglion cells of an entire regenerated retina. It is also evident from their work that some form of interaction may occur between the anterior and posterior parts of the eye during the regenerative process. The experiments in the present paper were undertaken with the following objectives in mind. (a) First, we intended to repeat the experiments of Burgen and Grafstein, in Triturus cristatus, in the hope of confirming and perhaps extending their results. (b) Secondly, it was hoped that we would be able to localize, more effectively, the source of positional information in the anterior part of the eye of the adult newt. MATERIALS AND METHODS
(1) Experimental animals The newts used in these experiments were adult T. cristatus, obtained from a commercial dealer. The animals were kept in ordinary fish tanks which were filled with tap water and kept at room temperature. A constant supply of Tubifex worms was kept in the tanks as food for the experimental animals.
(2) Surgical procedures (a) Eye operations. Prior to surgery, anaesthesia was effected by immersion in 0.4 ~ MS 222 (Sandoz) for 4-10 min. The animal was then placed in a plasticine holder and immersed in Holtfreter's solution. All ocular surgery was performed with the animal submerged. In one group of animals an incision was made entirely around the globe, at some distance behind the limbus corneae (corneo-scleral junction). These operations were similar to those performed by Burgen and Grafstein 1. In a second group of animals, the incision was made along the limbus. All incisions were made using a capsuiotomy scissors. After the incision had been made, the retina and lens were removed from the dissected eye. Removal of the retina was facilitated by the fact that it tended to float free of the retinal pigment epithelium (PE), when the eye had been opened. Removal was completed by tearing the remaining retinal attachments at the optic disc and the ciliary margin. Occasionally, small pieces of retina would remain attached at these two sites after the removal procedure. Such remnants could
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Post-limbal Incision
Lens and Retina Removed
Anterior Part Rotated and Reattachedto Posterior Part
Limbol Incision
Fig. 1. Schematic diagram showing operations described in text. Note the ventral notch in the pupil and the dorsoventrally asymmetric band of pigment along the limbus. Both of these characteristics were used to determine the orientation of the anterior part of the eye, during surgery and postoperatively.
be easily seen and were removed with watchmaker's forceps or by suction through a micropipette. The lens was also removed. After lens and retina were removed, the anterior part of the eye was rotated 180 °, in the plane of the incision, and returned to the posterior part of the eye. These operations are shown diagrammatically in Fig. 1. A series of control operations was also done, in which the anterior part of the eye was not rotated. The terms limbal and post-limbal will be used to describe respectively, (1) operations where an incision has been made along the limbus and (2) operations where the incision was made some distance behind the limbus. Correspondingly, operations in which the anterior part of the eye has been rotated will be referred to as either limbal or post-limbal rotations, depending on where the incision to the eye was made. After the operation, each animal was allowed to recover, for 3 days, in a moist container kept at 10 °C. Animals were then transferred to their home tanks at room temperature. (b) Preparation of anirnalsfor recording. Before recording, animals were immobilized with 0.18-0.25 ml 4 ~ Flaxedil (gallamine), 1 h before surgery. Immediately prior to the operation, they were anaesthetized with 0.4 ~ MS 222, for 3 min. The animal's skull was exposed through a longitudinal incision in the overlying skin. A rectangular bone flap was made, over the tectum, using a dental drill and the flap was reflected, exposing the brain and meninges. A drop of mineral oil was placed on the exposed tissues. The dura and subdural membranes were torn and reflected, exposing the tectum for recording.
REGENERATING RETINAL GANGLION CELLS IN THE NEWT
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Four animals, which had been subjected to post-limbal rotations, were recorded from between 3 and 4 months postoperatively. All other experimental animals were recorded from between 7 and 12 months after their operations.
(3) Recording procedures The microelectrodes used in this study were Woods metal filled micropipettes, Gestland et al. 5, with tip diameters of 2-5 btm. The electrode tips were plated with a ball of platinum black immediately prior to recording. The earth electrode was a stainless steel hypodermic needle which was inserted in the tail of the animal. The input stage of the amplification system was a Transidyne MPA-6 differential preamplifier which was used single ended, (input impedance of this device was 1011 f2). After suitable amplification, the signal was processed through a noise blanker which only passed pulses whose amplitude exceeded a preset level. From here the signal was relayed to an oscilloscope and to a loudspeaker.
(4) Mapping procedures After exposure of the optic tecta, the animal was placed on a cork platform and its eye was centred on the centre of a projection perimeter. The microelectrode was moved in 125 # m steps, across the surface of the tectum and the visual field position giving maximum responses at each tectal position, was noted. RESULTS
In normal animals, multiunit response field diameters ranged from 5-10 ° at the centre of the visual field to 15-20 ° in the periphery. In operated animals the response fields were consistently larger, usually ranging from 20 ° to 40 ° in diameter. In addition, in operated animals, there was no clearcut gradation of response field sizes, as one moved from the centre to the periphery of the visual field. The differences between response field sizes in normal and operated animals is presumably the result of abnormally extensive branching of the terminal arbors of the optic nerve fibres after retinal regenerationL However, because single unit recording was not done in the present experiments, one cannot say with certainty whether or not the enlarged multiunit response fields in operated animals were due, at least in part, to an increase in the size of individual single unit receptive fields. The normal VTP, of T. cristatus, is shown in Fig. 2. The visual field rows run ventrodorsally from their numbered extremities, which corresponds to a lateromedial progression of the points in the tectal rows. The most nasal portion of the visual field (row 1) is projected to the most anterior portion of the tectum, while the most temporal portion (row 8) is projected posteriorly. Only the dorsal part of the visual field is projected to the exposed portion of the tectum, the centre of the field being represented near its posterolateral pole. Studies in other species 1° indicate that the remainder of the visual field projects to the lateroventral, unexposed portion of the rectum. In animals that had undergone limbal or post-limbal operations, without rota-
324
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LEVINE
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CRONLY-DILLON
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tion o f the anterior p a r t o f the eye, the p o l a r i t y o f the regenerated VTP was always n o r m a l . However, if the a n t e r i o r p a r t o f the eye h a d been rotated, the VTP was always a b n o r m a l . Since animals with r o t a t i o n o f the a n t e r i o r p a r t o f the eye gave similar results regardless o f whether a limbal or p o s t - l i m b a l incision h a d been used, these results will be described initially w i t h o u t reference to the type o f incision. T w o m a j o r classes o f results were f o u n d in animals with the a n t e r i o r p a r t o f the eye rotated. A n example o f the first class o f results is shown in Fig. 3. N o t e that the field rows run d o r s o v e n t r a l l y in the visual field. Also, the most nasal field row D
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REGENERATING RETINAL GANGLION CELLS IN THE NEWT
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Fig. 4. Visuotectal projection of an animal in which a limbal rotation was performed 10 months previously. Note that there is a dorsal portion of the visual field which projects to the tectum with normal polarity (open circles). The ventral and lateral portions of the visual field project to the tectum with rotated polarity (filled circles). Rows 4 and 8, on the tectal surface, each receive input from two field rows in the visual field. Points on the tectum, which receive input from two loci in the visual field, are represented by horizontally divided circles. Some field rows in the rotated portion of the map are separated, on the surface of the tectum and in the visual field, into two segments, by the unrotated portion of the map (see rows 2 and 3). In these cases, the most medial segment on the tectal surface is denoted by an asterisk.
(row 8) projects to the p o s t e r i o r p a r t o f the tectum while the t e m p o r a l pole o f the visual field (row 1) is referred to the a n t e r i o r p a r t o f the tectum. These c o n d i t i o n s are the inverse o f those seen in the n o r m a l a n i m a l (see Fig. 2). Because o f this inversion, there is n o w a sizeable p o r t i o n o f the ventral visual field which projects to the d o r s a l surface o f the tectum. I n some a n i m a l s the a n t e r i o r p a r t o f the eye was r o t a t e d slightly m o r e or less t h a n 180 ° . I n these cases the visual field r e p r e s e n t a t i o n deviates f r o m a full 180 ° r o t a t i o n b y an a m o u n t c o m m e n s u r a t e with the e r r o r in the surgical r o t a t i o n . T h e field rows in m a p s which show c o m p l e t e inversion o f the V T P are generally well o r d e r e d t o p o g r a p h i c a l l y relative to each o t h e r a n d show little o v e r l a p with a d j a c e n t rows. T h e second class o f results f o u n d also in cases where the a n t e r i o r p a r t o f the eye h a d been rotated, consisted o f m a p s in which the visual field p r o j e c t i o n to the t e c t u m has divided into two t o p o g r a p h i c a l l y distinguishable parts. A n e x a m p l e o f this is seen in Fig. 4. The peripheral portions o f ventral, nasal, a n d t e m p o r a l areas o f the visual field in this a n i m a l are seen to project to the tectum with r o t a t e d polarity. I n the d o r s a l region o f the visual field, however, there is an area which projects with n o r m a l p o l a r i t y to the tectum. This a r e a fits into the space between the nasal a n d t e m p o r a l limbs o f the r o t a t e d p a r t o f the VTP. I t is n o t e w o r t h y t h a t we f o u n d no cases in the visual field where the two parts o f the m a p were superimposed. Indeed, the two m a p s a p p e a r to be m u t u a l l y exclusive in the visual field. H o w e v e r , one o c c a s i o n a l l y finds a slight o v e r l a p o f the field p r o j e c t i o n s on the tectal surface. This
326
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Fig. 5. Visuotectal projection o f an animal in which a post-limbal rotation was performed 7 m o n t h s previously. Note that there is a central, unrotated portion and a peripheral, rotated portion to the visual field map. Symbols as in Fig. 4.
can be seen, in Fig. 4, for those tectal portions represented by the horizontally divided circles. Each divided circle on the tectal surface represents a recording point which received its input from two areas, in the visual field. One of these areas was always part of the rotated projection and the other was part of the normal map. This is most clearly seen in visual field rows 8 and 8', which are widely separated in the visual field but project to the same row of recording points on the tectum. Another example of a two-part map is shown in Fig. 5. In this map the normal portion is centrally located and is almost completely surrounded by a peripheral band of the visual field which projects to the tectum with rotated polarity. This type of result was seen to occur in animals that had been subjected to post-limbal rotations. As in the previous example, the two parts of the map are mutually exclusive in the visual field and show only slight overlap on the tectal surface. In both types of two part maps, i.e., those which display a dorsally displaced normal portion as distinct from those with a normal portion that is located in the central part of the visual field, the field rows of the rotated map tend to be arranged topographically in an orderly fashion, except in the ventral part of the field where some overlap of rows may occur. The field rows of the normally projecting part of the map are often less regularly arranged than those of the rotated map, and even when they are regularly disposed with respect to one another, they are often distorted at the boundary which divides them from the rotated portion of the map (see Fig. 4). In no case did we observe a complete map with normal polarity and ordering, in animals that had undergone limbal or post-limbal operations where the anterior part of the eye was rotated. Recordings were made from 16 animals that had undergone post-limbal rotations. Of these, 8 showed complete rotation of the VTP, while another 8 gave two-part maps. Of the latter, 3 had dorsally displaced, portions of the visual field that projected
327
REGENERATING RETINAL GANGLION CELLS IN THE NEWT TABLE I
Operations
Post-limbal Limbal
Total number of animals
16 13
Types of maps recorded Complete rotation
8 3
Two part maps Dorsal normal portion
Central normal portion
Other
3 7
4 --
l* 3
* T h e m a p p i n g p r o c e d u r e was n o t c o m p l e t e d in this a n i m a l a n d the disposition o f the n o r m a l p o r t i o n o f t h e m a p c a n n o t be ascertained f r o m the d a t a which were recorded.
normally, while in 4 animals the normal projection was centrally located. In 1 animal the map we obtained was not complete enough to enable us to determine the disposition of the normal field. Of 13 animals with limbal rotations, only 3 showed completely rotated VTPs. The remaining 10 gave two-part maps of which 8 had dorsally displaced, normal portions. In 3 of these 8 animals, the putative normal portion of the visual field contained systematic abnormalities. Of the 2 remaining animals, 1 gave a single normally oriented row which was displaced ventrally in the visual field, the other animal had a large dorsally displaced normal field which extended ventrally to bisect the rotated portion of the field. These results are summarised in Table I. DISCUSSION
The purpose of this study was to try to localize the structure, or structures, responsible for specifying regenerating retinal ganglion cells in the eye of the adult newt. In all cases where the anterior part of the eye was rotated through 180 °, some or all of the regenerated VTP was found to be rotated. This result was obtained regardless of whether the incision freeing the anterior part of the eye had been made at, or behind the limbus. Control animals, where the anterior part of the eye had been replaced in a normal orientation, showed VTPs with normal polarity. This indicates that inversions of the map seen when the anterior part of the eye was rotated were not attributable to operative trauma. Thus, in animals that have undergone limbal or post-limbal rotations, the anterior part of the eye always appears to play a role in the specification of the regenerated retinal ganglion cells. Though many animals where the anterior part of the eye was rotated showed normally oriented portions of the VTP, none of them showed a completely unrotated map. In animals where we found two part maps, the normally projecting part was usually smaller than the rotated part and occasionally projected to only a portion of a single tectal row. These findings indicate that while the posterior part of the eye or the extraocular tissues may specify retinal cells, their 'specifying' effect on the entire regenerating retina is less predictable and not as strong as that of the anterior part. Since the unrotated source(s) of positional information exhibit such erratic
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behaviour in determining the polarity of the regenerating retina, one might expect such sources to be localized in a highly labile structure. The retinal PE is an obvious candidate for this role, since it is responsible for the regeneration of part of the retina and undergoes proliferative and metaplastic changes during this process7,1~-15. In addition, the extent of the retinal PE sheet initially remaining in the posterior part of the eye, varies from animal to animal, as a result of operative trauma 15. Thus the retinal PE, in the posterior part of the eye, is both labile and variable in extent and it is plausible to suggest that it may be responsible for specifying those cells in the retina that give rise to the normal portions of the two part maps. To test this hypothesis specifically would require one to repeat the present operations combined with removal of the PE from the posterior part of the eye. Such experiments are in progress and will be reported in a later paper. The simplest explanation for the two types of maps found in this study, i.e., two part maps and completely rotated maps, is that they represent varying amounts of growth of retinal tissue derived from the anterior, rotated part of the eye onto the posterior unrotated part. The completely rotated maps would then represent cases where the entire regenerated retina was derived from the anterior, rotated portion of the eye. If this explanation is the correct one, evidence of varying amounts of invasion of the posterior part of the eye by tissue derived from the anterior part should be available from histological studies. Indeed, histological data from the present experiments 15, indicate that in eyes that have been subjected to post-limbal operations there is a good deal of growth of the unpigmented cells of the anterior part of the regenerate onto the posterior part of the eye. The unpigmented lamina continues to spread until it is arrested by contact with regenerating retina derived from the posterior PE. Because regenerating retina in the posterior part may be at various stages of development when contact between the two occurs, the extent of spreading of the unpigmented cells varies from one animal to the next. However, in no case was the tissue derived from the anterior part seen to cover the entire posterior surface of the eye. Thus, although these observations may account in part for the observed results, they do not explain the occurrence of totally rotated maps which were found in 50 ~ of the animals with post-limbal rotations. Histological evidence suggests that in limbal eyes the anterior part of the eye is no longer the dominant source of new retinal ganglion cells 15. In addition a limbal incision effectively removes any positional cues, present in the retinal PE, from the anterior part of the eye. The occurrence of rotated visuotectal projections from such eyes therefore implies that some of the positional information imparted to the regenerating retina must derive from the regenerative tissues of the iris. In those animals, subjected to limbal rotations, which show completely rotated VTPs, the anterior part of the eye has specified an entire set of retinal ganglion cells, the majority of which presumably are derived from the unrotated retinal PE l~. Thus, the influence of the anterior part of the eye, on the specification of regenerating retinal ganglion cells, would appear to be able to extend into tissue not derived from the anterior part of the eye.
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Thus, there a p p e a r to be two i n d e p e n d e n t sources o f p o s i t i o n a l i n f o r m a t i o n in the eye o f the a d u l t newt. One source t h a t is l o c a t e d in the a n t e r i o r p a r t o f the eye, p r e s u m a b l y in the iris. P o s i t i o n a l i n f o r m a t i o n derived f r o m this region a p p e a r s to be c a p a b l e o f overriding a n y other p o s i t i o n a l i n f o r m a t i o n present in the eye. The second source is l o c a t e d in the p o s t e r i o r p a r t o f the eye, p e r h a p s in the retinal p i g m e n t epithelium. P o s i t i o n a l i n f o r m a t i o n derived f r o m the latter seems less stable t h a n t h a t derived f r o m the a n t e r i o r p a r t o f the eye a n d m a y indeed be o v e r r i d d e n b y i n f o r m a t i o n issuing f r o m t h a t part. ACKNOWLEDGEMENTS This research was s u b m i t t e d to the D e p a r t m e n t o f Biology b y R. L. Levine at Yale U n i v e r s i t y in p a r t i a l fulfillment o f the requirements for a Ph.D. degree. One o f us (R. L. L.) was s u p p o r t e d d u r i n g this research b y U S P H S Traineeship 5T01 HD-00032-09.
REFERENCES 1 BURGEN,A. S. V., AND GRAFSTEIN,B., Retinotectal connections after retinal regeneration, Nature (Lond.), 196 (1962) 898-899. 2 CRONLY-DILLON,J. R., Pattern of retinotectal connections after retinal regeneration, J. Neurophysiol., 31 0968) 410-418. 3 GAZE, R. M., The Formation of Nerve Connections, Academic Press, New York, 1970. 4 GEORGE,S. A., Studies on Optic Nerve Terminal Arborizations in the Frog's Tectum, Ph.D. Thesis, Johns Hopkins Univ., Baltimore, Md., 1970. 5 GESTLAND,R. C., HOWLAND,B., LETTVIN,J. Y., ANDPITTS,W. H., Comments on microelectrodes, Proc. Inst. Radio Engrs, 47 (1959) 1856-1862. 6 GRAFSTEIN,B., AND BURGEN,A. S. V., Pattern of optic nerve connections following retinal regeneration. In W. BARGMANNAND J. P. SCHADI~(Eds.), Topics in Basic Neurology, Progress in Brain Research, Vol. 6, Elsevier, Amsterdam, 1964, pp. 126-138. 7 HASEGAWA,M., Restitution of the eye after removal of the retina and lens in the newt, Triturus pyrrhogaster, Embryologia (Nagoya), 4 (1958) 1-32. 8 HUNT, R. K., ANDJACOBSON,M., Development and stability of positional information in Xenopus retinal ganglion cells, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 780-783. 9 HUNT, R. K., AND JACOBSON,M., Specification of positional information in retinal ganglion cells of Xenopus: Assays for analysis of the unspecified state, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 507-511. 10 JACOaSON,M., The representation of the retina on the optic tectum of the frog. Correlation between retinotectal magnification factor and retinal ganglion cell count, Quart. J. exp. Physiol., 47 (1962) 170-178. 11 JACOBSON,M., Retinal ganglion cells: specification of central connections in larval Xenopus laevis, Science, 155 (1967) 1106-1108. 12 JACOBSON,M., Development of neuronal specificity in retinal ganglion cells of Xenopus, Develop. Biol., 17 (1968) 202-218. 13 KEEFE, J. R., An analysis of urodelian retinal regeneration: I. Studies of the cellular source of retinal regeneration in Notophthalmus viridescens utilizing aH-thymidine and colchicine, J. exp. Zool., 184 (1973) 185-206. 14 KEEFE, J. R., An analysis of urodelian retinal regeneration: IV. Studies of the cellular source of retinal regeneration in Triturus cristatus carnifex using aH-thymidine, J. exp. Zool., 184 (1973) 239258. 15 LEVINE,R., Regeneration of the retina in the adult newt, Triturus cristatus, after surgical division of the eye, In preparation. 16 MATURANA,H. R., LETTVIN,J. Y., McCULLOCH, W. S., AND PITTS,W. H., Anatomy and physiology of vision in the frog (Rana pipiens), J. gen. Physiol., Suppl. 43 (1960) 129-175.