/. Embryo/, exp. Morph. Vol. 45, pp. 249-270, 1978
Printed in Great Britain © Company of Biologists Limited 1978
249
Anatomical mapping of retino-tectal
connections in developing and metamorphosed
Xenopus: evidence for changing connections
By ALISON LONGLEY 1
From the Department of Biology, University of Oregon, U.S.A.
SUMMARY
Neural connections between the eye and optic tectum in Xenopus laevis were anatomically
traced by observing the tectal location of Wallerian degeneration after discrete retinal lesion.
These retinotectal connections were mapped in postmetamorphic frogs and tadpoles at
stage 51, the stage at which retinal axons have grown into about the rostral one-half of the
tectum. The course of the experimental degeneration was the same in frogs and tadpoles,
but degeneration proceeded faster in the younger animals.
In the frogs, connections were ordered, with nasal retina mapping to the caudal part of
the tectum and temporal retina mapping to the rostral tectum. In the tadpoles, within the
innervated area at the rostral tectum, the retino-tectal connections were generally organized
as in the adults, with the temporal retina mapping to the rostral part of the innervated tectum
and nasal retina mapping primarily to the caudal part. But a portion of the nasal fibers
consistently mapped to the far rostral tectum as well. Electron microscopic observations
showed degenerating synaptic terminals at both rostral and caudal portions of the innervated
tectum after lesion of just the nasal retina. Degeneration was not seen in control animals.
These results indicate that some fibers (particularly from nasal retina) may shift their
terminals caudally on the tectum to match tectal growth and produce the adult pattern of
connections. If there is such connection readjustment, the 'aberrant' connections from nasal
retina in tadpoles may be an indication of this process.
INTRODUCTION
An explanation of the mechanisms by which nerve cells make and maintain
connections is fundamental to an understanding of the organization of the
nervous system. Several hypotheses have been proposed to explain the ability
of some neurons and sets of neurons to make specific contracts. The mechanisms
proposed include mechanical guidance (Weiss, 1955), differential timing of
axon growth (Jacobson, 1970) and chemospecific affinity of complementary
neurons (Sperry, 1963, 1965).
Of all the systems in which there is evidence for specificity in the formation
of connections between nerve cells, the visual system of lower vertebrates is one
of the most thoroughly studied. One reason is that there is a clear topographical
1
Author's address: Department of Zoology NJ-15, University of Washington, Seattle,
WA 98195, U.S.A.
250
A. LONGLEY
relationship between the position of retinal neurons and their connections in
the optic lobe, or tectum, making analysis of experimental perturbations of the
system relatively simple. Although embryologically part of the central nervous
system, the retina is uniquely accessible to experimental manipulation, and in
animals such as the amphibians Rana and Xenopus, the system can be manipulated even in the earliest embryonic stages, allowing the study of developmental
processes.
Several recent studies have been made of the retino-tectal connections as they
form during normal development. Connections of the entire eye have been
mapped histologically during development in Xenopus laevis (Scott, 1974;
Longley, 1974; Scott & Lazar, 1976; Jacobson, 1977); and Rana (Currie &
Cowan, 1975), and the visuo-tectal projection of Xenopus tadpoles has been
mapped electrophysiologically (Gaze, Chung & Keating, 1972; Gaze, Keating &
Chung, 1974; Chung, Keating & Bliss, 1974). It is evident from these studies
that during development in the frog, axons of the retinal ganglion cells from all
parts of the existing retina grow down the optic nerve to reach the antero-lateral
tectum at about stage 40. The retinal axons from the growing eye spread posteromedially to cover the entire tectum by about stage 64, shortly before metamorphosis at stage 66. Only electrophysiological methods have thus far been
used to map connections of parts of the retina (as opposed to the entire eye)
during development. Some of the potential difficulties in interpretation of
electrophysiological data have been pointed out (Hunt & Jacobson, 1974). In
the work described here, the location of degeneration in the tectum is determined
histologically after lesion of part of the retina. The retino-tectal connections of
larval and adult Xenopus are examined. Although there are inherent limitations
in both histological and electrophysiological methods, the retino-tectal relationships obtained using these methods are complementary in elucidating the
pattern of connections which are actually made as the axons of the retinal
ganglion cells innervate the optic tectum.
MATERIALS AND METHODS
Animals and operations
Adult Xenopus laevis for breeding were obtained from Jay E. Cook, importer,
Cockeysville, Md., and from MogulEd, Oshkosh, Wis. Methods of staging
animals, obtaining eggs and rearing larvae were as described in Nieuwkoop &
Faber (1956). Animals were kept at 18 °C except as otherwise indicated.
Juvenile frogs (stage 66) were at least 1 month post-metamorphosis and 2-33-5 cm in length (nose-rump).
Retino-tectal connections were mapped anatomically by observing the location of degeneration following lesion of a portion of the retina. The events and
time-course of this Wallerian (anterograde, orthograde) degeneration were
followed with light and electron microscopy in the stages to be mapped, and
Retino-tectal connections in Xenopus
251
the optimum degeneration times for mapping after retinal lesion were determined. The stages used were tadpoles at the early hind limb-bud stage 51, and
postmetamorphic juvenile frogs at stage 66.
For purposes of the degeneration study, the entire eye was removed after
cutting the optic nerve just behind the eye. To make focal lesions for mapping,
the skin near the eye was slit to expose the sclera and the eye was turned in its
orbit. Using fine forceps and iridectomy scissors, and cutting through all layers
of the eye, a portion of the eye was removed central to the iris. The eye was then
released and allowed to return to its normal position within the orbit. In
several animals, both eyes were lesioned and the two tecta could be directly
compared in the same animal. The anesthetic for operations was MS-222
(Sandoz) at a dilution of 1/3000 for larvae and 1/2000 for juveniles. After eye
removal or lesion, the animals were allowed to recover from the operation and
were kept at room temperature (24 °C) for a specified period of degeneration;
they were again anesthetized, and the brains were removed andfixedin 1 % (w/v)
OsO4 in amphibian Ringer's (Rugh, 1962). Tadpole brains were fixed 90 rain
at 0-4 °C; postmetamorphic brains were fixed for 30 min, then split at the
midline and fixed 90 min longer. They were then embedded in Epon (Luft, 1961;
Pease, 1964) and cut in cross-section on an ultramicrotome.
Microscopy
Thin sections for electron microscopy were stained with uranyl acetate (5 %
in HaO) and with lead citrate (Reynolds, 1963). Staining procedures were as
described in Pease (1964) except for mass staining of grids, which utilized the
method described by Sjostrom, Thornell & Hellstrom (1973). For light microscopy, the same plastic embedded tissue was used as for electron microscopy,
but sections were cut 1 /tm thick.
Mapping procedure
Serial 1 jam cross-sections were taken through the optic tecta and examined
with the phase optics of a Zeiss microscope at 200 x .
An eyepiece micrometer with a square grid containing 20 x 20 squares was
used to construct sketches of the location of black dots indicating degeneration
in relation to the tectal outline (Fig. IB). The grid squares were 16-3/im on a
side. In scoring degeneration (seen as black dots using phase microscopy at
200 x magnification) a criterion of at least three dots per small square for
tadpoles and four dots per square for adults was set. These criteria were set at
a level low enough to include most areas of known degeneration (i.e. innervated
tectum after whole eye removal), and large enough to exclude most areas of
control tecta and thus avoid spurious counts from blood vessels, etc. Before
scoring for degeneration, slides were shuffled randomly and assigned code
numbers in a single-blind manner to eliminate any possible bias in scoring.
1
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Retino-tectal connections in Xenopus
253
Two-dimensional maps of degeneration were constructed from the sketches of
the tectal cross-sections (Fig. 1).
Preliminary experiments indicated that in tadpoles, eye lesions which were
smaller than about 0-6 mm in diameter gave degeneration that was insufficient
for mapping. For that reason, the tadpoles were fixed and embedded in paraffin
after the brains had been removed. Serial frontal sections (10 jum) were taken
through the anterior end of the animal to include the eyes. The sections were
stained with Ehrlich's acid alum hematoxylin (Gray, 1954). Eye lesion diameter
was determined from these sections. Tadpoles were used for mapping in which
the lesion was confined to the appropriate quadrant with no apparent injury
to the rest of the eye and in which the lesion was between 0-6 and 0-95 mm in
diameter. The larger size of the juvenile eyes made the lesion operation easier,
and all of the juveniles were used for mapping.
RESULTS
Selection of degeneration times
The optimum times for mapping degeneration in the tectum following retinal
lesion were determined by following the time-course of degeneration with light
and electron microscopy. The course of degeneration is very similar in stage-51
tadpoles and metamorphosed frogs, but proceeds much more quickly in the
tadpoles. The first signs of degeneration seen ultrastructurally are osmiophilic
bodies the size of mitochondria in synaptic terminals at 4 and 8 h in tadpoles
and 1 day in juveniles (Fig. 2 A). The debris appears to arise from clumped
synaptic vesicles and mitochondria (Fig. 2B, C). By 16 h in tadpoles and 4 days
in juvenile frogs, ependymal glia begin to envelop the terminal debris (Fig. 3 A, B),
and this ependymal debris is prevalent at 20 and 24 h in tadpoles; 4 and 5 days
Fig. 1. Procedure for making two-dimensional maps of tectal degeneration from tectal
cross-sections. Sketches of the location of spots of degeneration were made (B) from
tectal cross-sections (A). The x s marking degeneration were located in two dimensions; latero-medial and rostro-caudal. The lateral dimension is expressed as degrees
of arc along the curved surface of the tectum in cross-section, with 0" at the midline
(B). For these measurements, the origin of the protractor is set on the midline at a
distance 2 dfrom the tectal surface, where d is the distance along the midline from the
tectal surface to the point where the lateral extent of the ventricle is greatest (D).
The longitudinal dimension is the position of the section along the length of the
tectum (C), expressed as percentage of the total tectal distance with 0 % at the
rostral end. Reference points for location along the longitudinal axis are, in frogs,
the point of greatest width of the ventricle (at 70 % of the total tectal distance, (D),
and in tadpoles, at the point where the ventricle becomes three-Iobed (at 90 % of the
total distance; E). (F) shows a completed map of areas of tectal degeneration in
which the degeneration areas of (B) form a horizontal row of small rectangles
to the left of the arrow. Each rectangle on the map represents one or more of
the small squares of the eyepiece grid (A) which contained degeneration sufficient
to meet the criterion (i.e. three black spots/square for this stage-51 tadpole).
17
EMB 45
254
A. LONGLEY
Fig. 2. (A) Electron micrograph from a stage-51 tadpole tectum 8 h after section of the
optic nerve. Two nerve processes with synapses (S) and synaptic vesicles (SV) also
contain osmiophilic debris (DB) bounded by a membrane. Bar represents 1 pm.
(B) Clumped vesicles bounded by a membrane (CSV) 8 h after nerve section. The
clump contains both spherical and dense-core vesicles. Stage-51 tadpole. (C) An
abnormal mitochondrion (M) near a synaptic cleft with vesicles (SV) 8 h after optic
nerve section. Note darkening of the cristae. Stage-51 tadpole.
Fig. 3. (A) An ependymal glial cell containing debris (ED). Ultrastructural view at the tectal surface (S) of a stage-51 tadpole 24 h
after optic nerve section. (B) Ependymal glial debris (ED) in the juvenile tectum after 4 days of degeneration. The debris is patchy,
dark, and variable in size.
X
GO
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d
P
O
256
A. LONGLEY
Fig. 4. (A) Phase micrograph of a tadpole tectum in transverse section at stage 51
one day after section of the optic nerve. The tectal surface is at the top. Numerous
small black dots (D) are located outside of cell bodies in the outer layer of the tectum.
Nucleoli (not counted in maps) can be distinguished from these spots by their location within cell bodies (CB). (B) Transverse section from the tectum of a control
stage-51 tadpole. Note the relative absence of black dots. (C) Stage-51 tadpole
tectum in transverse section 3 days after optic nerve section. Note the change in
distribution and the increased size and density of the black dots.
in frogs. Numerous small black dots in the optic layer of the tectum can be seen
with light microscopy beginning at about 20 h after nerve cut in the tadpoles
and 4 days in the juvenile frogs. Their maximum number is reached at about
24 h and 5 days (Fig. 4). Comparison of neighboring thin and thick sections
with light and electron microscopy demonstrated correspondence of the
ependymal debris with black dots seen with light microscopy (Fig. 5 A, B). The
times following eye lesion which were selected for mapping the tectal debris of
degeneration were 1 day for tadpoles and 5 days for juvenile frogs.
Postmetamorphic juveniles
Removing the entire retina in juveniles (eight animals) results in the appearance of degeneration over the entire contralateral tectum (Figs. 6 and 7). There
is some variation between individuals in density of degeneration, which is
Retino-tectal connections in Xenopus
257
Fig. 5. (A, B) Subadjacent phase and electron micrographs of the frog tectum after
4 days of degeneration. A blood vessel (BV) and the tectal surface (S) serve as landmarks. The osmiophilic debris in B is contained in electron-lucent cytoplasm
characteristic of ependymal glia. (Shown at greater magnification in Fig. 3B.)
Some of the phase-dark dots and ultrastructural debris which appear to be in
correspondence are numbered.
258
A. LONGLEY
Optic tectum
Fig. 6. Map of tectal degeneration following retinal lesion in a juvenile frog. Direct
retinal connections end in the contralateral tectum. The type of retinal lesion is
indicated by a blacked-in area; in this case, the right eye was removed completely,
and a lesion was made within the temporal quadrant of the left eye. The location of
the lesion-induced degeneration is indicated by the rectangles on the tectum. The
maps were constructed as described in Fig. 1 and the text.
% total tectal length
s
OS
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3
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~
X
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Caudal
3
Rostral
O
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260
A. LONGLEY
Optic tectum
100
80
60
90' I •*
40
20
Fig. 8. Map of retino-tectal connections in a frog. No lesion was made in the right
eye. Conventions as in Fig. 6.
Retino-tectal connections in Xenopus
261
artificially enhanced in the maps because of the use of a density criterion for
scoring degeneration. The one unoperated control animal showed no degeneration (Fig. 8).
The temporal retina maps to the rostral tectum in all of the seven animals
examined. The nasal retina maps to the caudal tectum in six of the eight frogs,
with a small scattering of degeneration also appearing at the rostral portion of
the tectum in three of the six. Two frogs showed very little degeneration, perhaps
because the eye lesion was too small. Figures 6-8 show representative retinotectal projections of nasal and temporal retina in juvenile frogs.
The mapping results with temporal lesions and entire retinal removal are
consistent with previously published electrophysiological studies on adult
Xenopus (Gaze, 1958; Gaze, Jacobson & Szekeley 1963; Jacobson, 1968) and
histological studies of Triturus and Rana (Stroer, 1939; Lazar, 1971; Scalia &
Fite, 1974). The previously published data also suggested that the nasal retina
projects solely to the caudal tectum, but in the present experiments, a portion
of the nasal fibers projected to the rostral tectum, though most did in fact project
caudally. The apparent degeneration at the rostral tectum may be simply due
to spurious counts, or this difference of projection may be a function of the
maturity of the animals. The frogs used in the present experiments were
2-3-3-5 cm in length and from 1 to about 5 months postmetamorphosis, while
those used in other studies have generally been much larger and older.
Tadpoles
After removal of the entire eye in six tadpoles, the ensuing degeneration
appeared at the rostral part of the tectum (Fig. 9). The caudal extent of the
degeneration varied with the individual animals from about 45 % to 85 % of
the total tectal length. Degeneration was more extensive at the rostral part of
the innervated tectum. Areas of the tectum which were thin and not yet well
laminated (the far caudal tectum) showed practically no degeneration (Fig. 9).
It was consistently found that unoperated control animals showed very little
or no tectal degeneration. Of the four unoperated controls, two showed no
degeneration and two scored for degeneration as shown in Fig. 10. Degeneration
in unoperated animals has been reported in stage-45 Xenopus, decreasing by
stage 47 (Scott, 1974). Periods of degeneration in developing nerve centres are
known to be a part of normal development in several systems (Jacobson, 1970;
Cowan, 1973; Rogers & Cowan, 1973), but by stage 51 in Xenopus, degeneration
is virtually nil in control maps.
Of the eight animals in which a lesion of the temporal retina was made, six
mapped completely, or nearly so, to the far rostral tectum (Figs. 11, 12). One
showed no degeneration, and one showed degeneration at both rostral and
caudal parts of the tectum (Fig. 13). The temporal retina of the tadpoles mapped
closer to the rostral pole of the tectum than it did in juveniles with comparable
retinal lesions. In contrast to the pattern of tectal degeneration that is evident
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Retino-tectal connections in Xenopus
Optic tectum
Fig. 10. An unlesioned control tadpole of stage 51. Cross-hatched area indicates an
eye and tectum which were not mapped. Conventions as in Fig. 6.
263
3
3
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5'
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Temporal
Temporal
Rostral
"o total tectal length
Caudal
4
to
Retino-tectal connections in Xenopus
Optic tecturn
100
80 +
60
0°
40
20' •
0;
Fig. 12. Map of retino-tectal connections at stage 51. Conventions as in Fig. 6.
265
266
A. LONGLEY
Optic tectum
100
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80 -
PA
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I
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90°
60°
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60'
30°
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Fig. 13. Map of retino-tectal connections at stage 51. The caudal tectal degeneration
following temporal retinal lesion is unusual. Conventions as in Fig. 6.
Retino-tectal connections in Xenopus
267
Figs. 14 and 15. Electron micrographs of degeneration in synaptic processes. These
sections were taken from the rostral portion of the innervated tectum in tadpoles
at stage 51 following lesion of the nasal retina. Synapses are marked by arrows;
debris characteristic of degeneration (D) is close by. Silver to grey sections poststained with lead citrate and uranyl acetate.
after lesion of the temporal retina, and despite similar eye lesions, degeneration
in the tectum after lesion of the nasal retina in tadpoles was widespread. Of the
nine animals in this category, one had debris localized almost entirely at the
caudal part of the innervated tectum (Fig. 1F), one showed virtually no degeneration, and seven showed extensive degeneration at both rostral and caudal parts
of the innervated tectum. In those seven animals, the greater amount of degeneration was at the caudal part (Figs. 9, 11, 12, 13).
Because debris of degeneration at the rostral tectum after nasal eye lesion
could conceivably be due to axonal rather than terminal degeneration (the nasal
retinal ganglion cell axons cross over the rostral tectum to terminate at the
caudal tectum in adults), a study of the ultrastructural location of the degeneration was undertaken. Nasal retinal lesions were made in both eyes of several
tadpoles. The lesion of the left eye was made 24 h before fixation (the time used
for mapping the black dots with light microscopy), and that of the right, 8 h
before (the time at which debris is observed ultrastructurally in synaptic
terminals). This procedure produced animals in which early and late stages of
degeneration were present on different sides of the same brain. The brains were
fixed, and the eyes were fixed and sectioned as described earlier. Only animals
in which both eyes met the lesion criteria mentioned previously were examined
further. Thick sections of the optic tectum were examined with the light micro-
268
A. LONGLEY
scope for degeneration of the 24 h side at the rostral portion of the innervated
tectum (15-35 % of the total tectal distance). Sections which showed black dots
in that region were remounted for sectioning (Schabtach & Parkening, 1974),
trimmed to cut away the 24 h side (the time at which the degeneration is seen
with light microscopy) leaving only the 8 h side (the time at which degeneration
can be seen ultrastructurally in synaptic terminals) and sectioned for electron
microscopy.
Thin sections from the rostral 15 % to 35 % of the tecta of two animals contained synaptic processes with debris (Fig. 14, 15). Quantification of the debris
of degeneration was not attempted, but the debris did appear to be sufficiently
prevalent to account for the degeneration seen with the light microscope at the
rostral tectum after nasal retinal lesion. Sections from the caudal innervated
tectum at 50-70 % of the entire tectal distance were also examined for degenerating synapses and they were found, as expected. These results show that
fibres from the nasal retina have synapses at both rostral and caudal parts of
the innervated tectum at stage 51.
DISCUSSION
Wallerian degeneration can follow one of a number of courses, depending
on the tract and the species (Raisman & Matthews, 1972). The degeneration
found here in retinal fibres after they have been severed from their cell bodies
is similar in its first stages (the production of dark debris in otherwise healthylooking terminals) to that reported in cats (Hamori, Lang & Simon, 1968). The
subsequent engulfment of this debris by ependymal glia has also been reported
in other amphibian visual tracts (Scott, 1973, 1974; Reier & Webster, 1974;
Turner & Singer, 1975). The glial engulfment produces debris large enough to be
mapped with light microscopy.
Wallerian degeneration in some systems is known to begin at the terminal
end of the severed axon, and axonal degeneration proceeds later (Hamori et al.
1968). Relatively short degeneration times were chosen here to minimize the
possibility of axonal degeneration, but in practice it is not possible to be certain
that all of the debris mapped with light microscopy is terminal. Where necessary
for interpretation of the maps, the source of the degeneration in particular areas
can be checked with electron microscopy, as was done here for degeneration
appearing in both rostral and caudal tectum after lesion of the nasal retina in
tadpoles. The finding of terminal degeneration in both tectal areas demonstrates
that ganglion cell bodies located in the nasal retina have at least some terminations in rostral as well as caudal tectum during their period of axon outgrowth.
To summarize, fibres from both temporal and nasal retina terminate in the
optic tectum even at an early stage of innervation. The retinotectal map at this
stage is organized roughly like an adult map in miniature on the innervated
portion of the tectum. This is in agreement with the electrophysiologically
Retino-tectal connections in Xenopus
269
obtained results of Gaze et af. (1972, 1974), and indicates that some fibres
(particularly from nasal retina) may shift their terminals caudally on the tectum
to match tectal growth in order to produce the adult pattern of connections
(Gaze, 1974). Substantial numbers of connections which are 'aberrant' in the
sense that they are not located in accordance with the adult projection are
present in the stage-51 tadpole. Most of the aberrant terminals come from the
nasal retina, which forms terminals in the rostral, as well as the expected
caudal, tectum. The incorrect connections appear to be considerably reduced
in number in postmetamorphic juveniles, although it is possible that the aberrant
connections are retained, but are 'diluted' by the addition of large numbers of
correctly made synapses. Gaze et at. (1972, 1974) observed that in stage-47
tadpoles, the receptive field of electrodes placed in the rostral tectum is much
larger than that of more caudally placed electrodes. This distinctive difference
in receptive field disappears as development proceeds. As noted by Gaze (1975),
these observations both suggest that there is a reduction in the relative number
of incorrect connections with development. If there is readjustment of retinal
connections as the tectum grows, the 'aberrant' connections observed here in
the tadpoles at stage 51 may be an indication of this process.
1 would like to thank Dr Philip Grant and Dr Edith Maynard for stimulating discussions
and helpful criticisms during the course of this work, and Dr J. S. Edwards and Dr R. D.
Lund for comments on the manuscript. This research fulfills, in part, the requirements for
the degree, Doctor of Philosophy, and was supported by contracts AT (45-l)-20ll and
AT (45-0-2230 to Dr Philip Grant and Health Science Advancement Award FR-06027 from
the National Institutes of Health.
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{Received 11 November 1977, revised 19 December 1977)