/. Embryol. exp. Morph. Vol. 33, 4, pp. 915-940, 1975
915
Printed in Great Britain
The structural and functional development
of the retina in larval Xenopus
By S. H. CHUNG, 1 R. VICTORIA STIRLING 1 AND R. M. GAZE 1
From The National Institute for Medical Research, Mill Hill, London
With an Appendix by
M. LAND AND R. VICTORIA STIRLING
2
SUMMARY
The structural transformations of the larval Xenopus retina at successive stages of development, and concomitant changes in response characteristics of retinal ganglion cells, were
studied using histological and electrophysiological techniques. The first sign of visually
evoked electrical responses appears at about the time when the ganglion cells spread out into
a single layer and shortly after the inner and outer plexiform layers become discernible.
Initially giving simple 'on' responses, the cells progressively change their response characteristics and become 'event' units. Subsequently, 'dimming' units can be identified. Throughout larval life, response properties of these two types become more distinct from one another
and approximate to those found in the adult. So do the arborization patterns of the dendritic
trees of the ganglion cells. Two types of branching patterns are identifiable in Golgi preparations. Around metamorphic climax, a new type of ganglion cell appears, coinciding with
the emergence of 'sustained' units electrophysiologically. After metamorphosis, the retina
still grows both in thickness (mainly in the inner plexiform layer) and diameter. The three
unit types change such that they come to show pronounced inhibitory effects from the
peripheral visual field on the receptive field and each unit type acquires a distinct pattern of
endogenous discharge.
INTRODUCTION
The development of the visual system in amphibians has recently received
considerable attention. Autoradiographic studies on the developing retina in
Xenopus have shown that cells are added continuously at the ciliary margin
throughout larval life and until after metamorphosis (Straznicky & Gaze, 1971;
Hollyfield, 1971). The steady increase in the number of retinal cells and optic
nerve fibres (Gaze & Peters, 1961; Wilson, 1971) during larval life is accompanied
by a marked thickening of the retina and changes in the synaptic organization
in the inner plexiform layer (Fisher, 1972).
Since the function of a neuron is intimately related to its structure and its
connexions with other neurons, morphological changes in the developing
1
Authors' address: National Institute for Medical Research, Mill Hill, London NW7 1AA,
U.K.
2
Author's address: Sussex University, Falmer, Sussex, U.K.
57
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33
916
S. H. CHUNG AND OTHERS
retina will result in accompanying modification of the receptive field properties
of retinal ganglion cells. Pomeranz & Chung (1970) and Pomeranz (1972) have
noted that the receptive field properties of visual units do undergo changes
during amphibian metamorphosis, and these changes appeared to be related to
alterations in the shapes of the ganglion cell dendritic trees.
The present paper describes the development of form and function in retinal
ganglion cells in larval Xenopus. As in the previous studies (Pomeranz &
Chung, 1970; Pomeranz, 1972), receptive field properties of single retinal
ganglion cells were studied by recording from the terminal arborizations of their
optic nerve fibres in the tectum, and the structure of the developing retina was
examined using Holmes's silver stain and Golgi methods. A preliminary account
of some of these observations has been communicated elsewhere (Chung,
Gaze & Stirling, 1972).
METHODS
Preparation. Thirty-two adult Xenopus laevis, aged approximately 2 years,
and 156 tadpoles at various stages of development were studied. Adult toads
were anaesthetized with ether, decerebrated, and immobilized by injecting
0-5 mg D-tubocurarine into the dorsal lymph sac. The skull was opened over
the tectum, and the dura membrane covering it was carefully slit open. The
blood vessels lying on the surface of the Xenopus tectum are firmly affixed to a
thick pia, which cannot usually be removed without causing a major haemorrhage. This membrane was degraded enzymically in some animals to facilitate
the passage of the electrode tip. Trypsin, 5 %, in tris buffer of pH 7*6 was applied
to the surface of the tectum for 5-10 min and then washed away thoroughly
with saline. We could not discern any difference in the responses of units recorded from tecta treated with trypsin from those without trypsin. The brain
was then covered with a layer of mineral oil.
Tadpoles were first anaesthetized by brief immersion in 1:3000 MS 222
(tricaine methanesulphonate - Sandoz) and then immobilized after decerebration by injection of approximately 0-03 mg D-tubocurarine. The skin and
cartilage (in older tadpoles) over the tectum were removed with a pair of fine
forceps and the dura membrane was slit and pulled aside. The animal was
pinned on to a Plasticine platform such that one eye was at the centre of a
hemispherical Perspex dome 10 cm in diameter which was filled with oxygenated Niu Twitty solution (Wilt & Wessels, 1967). A hole 2-5 cm in diameter
at the top of the hemisphere permitted an indifferent electrode to be immersed
in the solution and the microelectrode to be placed on the tectum contralateral
to the centred eye. The hemisphere was placed at the centre of an 'Aimark'
perimeter (U.K. Optical - Bausch & Lomb). The distance between the eye of
the tadpole and the arc of the perimeter was 33 cm. Under this arrangement a
line of sight from any small visual stimulus always lay normal to the air-water
interface, thereby minimizing refractive distortion.
Development of retina in Xenopus
917
Since the rostrocaudal extent of the tadpole tectum shows different degrees of
maturity at any one stage of development (Straznicky & Gaze, 1972; Lazar,
1973), we have attempted to record unit activities from slightly to the temporal
side of the centre of the visual field. Animals were staged according to the table
of Nieuwkoop & Faber (1956).
Recordings. Electrodes were metal-filled glass pipettes tipped with gold and
platinum. The diameter of the platinum ball was 5-10 /im. Recordings were made
through a conventional low-noise a.c. amplifier of bandwidth 100-10000 Hz
and an input resistance of 10 MQ.. The impulses were displayed on an oscilloscope and were monitored over a loudspeaker. A device called a CLOOGE
(acronym for Continuous Log Of On-Going Events) was employed to display
a continuous plot of successive interspike intervals (Chung, Lettvin & Raymond,
1974). This device generates, for every triggering impulse supplied, a single
square wave of voltage whose amplitude is the logarithm of the time interval
from the preceding pulse. To generate the figures shown, the output of the
CLOOGE was fed into a second oscilloscope and the peak of this square wave
was brightened to give a dot. A slow sweep on the oscilloscope was, when needed,
obtained by a motor-driven potentiometer supplied by a constant-voltage
source.
Visual stimuli. The perimeter arc was uniformly illuminated by a 7 Watt
bulb. The luminance of the arc was 2 cdm~2, and this intensity is designated
as the ambient illumination throughout the paper. Attenuation of stimulus
intensity over three log units was achieved by a potentiometer placed in series
with the light source. A 2 ° spot of light of 20 cdmr 2 as well as black discs of
various diameters were also used as stimuli.
Physiological optics. The Xenopus adults and tadpoles were somewhat hypermetropic in water and emmetropic in air. The diameter of the blur circle for
tadpoles in water was approximately 6°, and remained constant throughout
larval life. The size of the blur circle in air will be smaller, as the cornea in air
becomes a refracting surface. Because of the difficulty in keeping the tadpoles
alive in air, and to approximate a more normal visual environment, we have,
nevertheless, recorded from tadpoles in Niu Twitty solution as described above.
For detailed discussion of the optics of Xenopus eyes, see Appendix.
Histological methods. Holmes's silver preparations were made on whole
tadpoles fixed in Susa and Carnoy, using standard procedures.
Various modifications of the Golgi rapid method were used. Best results were
obtained when eyes were injected with 1 part of 2 % osmium tetroxide to 4
parts of fresh potassium dichromate. The heads were left in this fixative for
3-4 days for stage 55-60 tadpoles and for 7-8 days for stage 48-53 animals,
followed by a similar time in 0-75 % silver nitrate. Impregnation was always
better in the older tadpoles; in younger tadpoles successful preparations were
made using a 5-10 min prefixation with 5 % glutaraldehyde in Niu Twitty
solution. After the usual dehydration procedures, the specimens were embedded
57-2
918
S. H. CHUNG AND OTHERS
D
Development of retina in Xenopus
919
in Necoloidine (BDH). Sections, cut horizontally or transversely at 80 /.im,
were cleared in 50 % chloroform in alcohol and then xylene before mounting
in DPX with coverslips.
RESULTS
/. The morphology of the developing retina
Histogenesis. The retina is first clearly identifiable in Holmes's silver stained
preparations slightly before stage 30. Both the lens and the retina consist of
masses of undifferentiated cells lying within a cup of pigmented epithelium.
The retina has a visible fault ventrally, probably representing the future exit
point of the optic nerve. Fig. 1A illustrates these structures in a stage-32 animal.
At stage 33 the retina, which is approximately 50 ju,m thick, shows the first
signs of differentiation with the appearance of darkly staining cells on the inner
margin towards the lens. This layer becomes more obvious at stage 35 (Fig. 1B)
when these cells have sent out axons which can be identified at the optic chiasma
(Fig. 1 C), but which have not yet reached the tectum. At this time a second
row of darker staining cells appears, situated just inside the pigment epithelium;
these cells are the future outer nuclear layer cells.
At stage 37 a cell-free band, 8-10 ju,m wide (the inner plexiform layer),
forms above the ganglion cell layer (which is still several cells thick) along the
middle third of the retina (Fig. ID). At this stage also the first sign of the outer
plexiform layer can be seen, separating the receptor cells from the inner nuclear
layer. Between stages 37 and 42, the inner plexiform layer expands to 15/*m
wide, and the receptor cells start to elongate. The lens is well formed and
acquires the adult crystalline structure at the end of this period. The lateral
ventricles of the optic tecta start to develop at this time, and the optic fibres
reach the brain, forming a fibrous layer above the more anterior tectal cells.
Subsequent development of the tectum is described in detail elsewhere (Lazar,
1973; Gaze, Keating & Chung, 1974).
FIGURE 1
Photomicrographs of larval Xenopus retinae and optic chiasma. Transverse sections,
Holmes's silver stain. Calibration for (A) and (B), 100 /*m; for (C), 50 /*m, and for
(D), 100 /im.
(A) The eye of a stage-32 Xenopus shows a conspicuous fault in the ventral aspect of
the eye-cup, representing the future exit point of the optic nerve.
(B) The ganglion cell layer (arrowed) can be seen near the centre of the retina from a
stage-35 tadpole.
(C) The optic chiasma can be identified, and optic fibres (arrowed) seen crossing, in
a stage-35 tadpole. This section is from the same animal as that illustrated in (B).
Ventral is to the left and dorsal to the right.
(D) The central part of the retina of a stage-37 tadpole shows a cell-free region, the
inner plexiform layer. The outer plexiform layer also begins to be visible at this stage.
The optic nerve can be seen leaving the eye ventrally (top in the photograph).
920
S. H. CHUNG AND OTHERS
80000
w
Fig. 2. Thickness of the outer (O) and inner ( # ) plexiform layers and the number of
optic nerve fibres ( x) as a function of age. The plexiform layers are measured near
the optic nerve head in Holmes's silver preparations from at least four animals in
each group. Corrections for shrinkage are not made, as the absolute values of thickness are subject to considerable error (see text). Fibre counts are plotted from the
data provided by Wilson (1971). The abscissa relates main phases and stages of
development with approximate age in days.
Retinal layers. By stage 45 the ganglion cells spread out into an ordered single
cell layer and the retina grows further in thickness, mainly in the plexiform
layers, until stage 53 (Fig. 2). It increases in diameter, by the addition of rings
of cells to all three layers at the ciliary margin, throughout larval life (Straznicky
& Gaze, 1971).
There appears to be very little mitosis in the fundus of the retina once the main
region of proliferation at the ciliary margin has moved away. Hollyfield (1971)
has stated that the inner nuclear layer undergoes an extensive increase late in
larval life, with increase in the cellular thickness of the layer from two to four
cells between stages 55 and 65. Our preparations do not support this conclusion. In the animals used in the present series of investigations, the inner
nuclear layer is found to vary in thickness (measured in all cases from the
fundus close to the optic nerve head in coronal sections) from animal to animal.
This layer is thickest (23 (im) at stages 45-52, while after stage 58 it is 18 /mi
Development of retina in Xenopus
921
VVJW1
Fig. 3. Transverse sections through the retinae of a stage-60 tadpole (left) and a 6
months post-metamorphic juvenile fright). Sections were taken in each case from a
comparable part of the fundus at the region of greatest circumference of the eye.
Holmes's silver stain. Calibration, 20/tm. (A) Ganglion cell layer; (B) inner plexiform layer; (C) inner nuclear layer; (D) outer plexiform layer.
thick. Whereas absolute layer thickness can be misleading in that it is dependent
on the vagaries of fixation and the cutting angle, this difficulty is not so great
when cellular thickness is estimated. The inner nuclear layer is consistently
found to be three or four cells thick in all regions from stage 45 up to adult life.
Although the thickness of the retina remains fairly constant from stage 53
to metamorphic climax, there is a steady increase in the numbers of optic nerve
fibres (Fig. 2) and of cells in all cellular layers. The growth of the retina continues after metamorphosis. Holmes's silver preparations show that adult
Xenopus aged from 6 months to 2 years after metamorphosis have retinae
922
S. H. C H U N G AND OTHERS
Fig. 4. Montage of camera lucida drawings of retinal ganglion cells. Drawings were
made from Golgi preparations. The dotted line demarcates the outer limit of the
inner plexiform layer. All cells except (C) were derived from the central portion of
the retina. Calibration, 100 /im. (A), (B) Two representative cells from a stage-48
tadpole. (C) Cell from the peripheral retina of a stage-51 tadpole. (D), (E) Two
patterns of dendritic arborizations from a stage-57 tadpole. (F), (G), (H) Single-tiered
(T-type) and two-tiered (H-type) cells from a stage-64 tadpole. (I), (J) Small bushytree cells from a stage-64 tadpole. Note that the dendritic fields occupy the middle
region of the inner plexiform layer.
considerably thicker than those at stage 60 (Fig. 3). The greater part of this
increase is due to the inner plexiform layer which, in the example shown,
changes from 20 to 35 /*m.
Dendritic trees. We have not been able to obtain proper Golgi impregnations
of cells before stage 47. The shapes of ganglion cell dendritic trees between
stages 48 and 53 show considerable variability. In the centre of these early
retinae the cells have branches extending over the whole depth of the inner
plexiform layer (Fig. 4A, B), whereas at the margin of the retinae where this
layer is narrower the dendritic spread is more restricted (Fig. 4C).
In animals from stages 54-60, the branching patterns of the ganglion cells
are more complex than those in younger tadpoles. Close to the retinal margin,
cells with broad arborization (resembling Fig. 4C) are occasionally found. Over
the rest of the retina there are two distinguishable types of cells (Ramon y
Cajal, 1911). The first type have arborizations at two levels, one tier just above
the cell body and the other extending laterally above them, just below the
inner nuclear layer (Fig. 4D). The second type of cells have only a single tiered
arborization, just below the inner nuclear layer (Fig. 4E).
Development of retina in Xenopus
g 20
Stage 47
Stage 49
Stage 55
923
Stage 59
a
on
O
10
o
a
in
2 10
~ 20
Fig. 5. Relative strength of 'on' and 'off' responses of 'event' units to punctiform
stimulation at different stages of development. The number of spikes elicited by on
and off of a spot is indicated above and below the baseline, respectively. The stimulus
was moved in 5° steps across the major axis of the receptivefields.Note that the 'off'
component emerges later in development than the ' on' component, and the size of
the receptive field decreases progressively.
From stages 61 to 66 the best impregnations are seen. At these stages the
amacrine processes also impregnate well and their denseness makes the identification of ganglion cell shapes sometimes difficult. Two-tier and single-tier cells
are present in approximately equal numbers (Fig. 4F-H), and a third type of
cell, not observed in younger tadpoles, is occasionally found. These cells have
denser bushy arborizations extending about half-way up the inner plexiform
layer with a dendritic field of 60 jura or less (Fig. 41, J).
//. The physiology of the developing retina
Stages 43-48. Stage 43 was the earliest period of development at which we
could reliably record visually evoked action potentials from terminals of optic
axons in the tectum. The tectal region from which activities could be recorded
was confined to the superficial layer at the rostrolateral pole. Units discharged in
a cluster of four to five spikes at the onset of a bright light, and this response
habituated quickly to repeated presentation of the stimulus. The size of the
receptive field could not be determined at this stage, as these units were unresponsive to a spot of light or a disc.
Between stages 43 and 48 the animal grows rapidly, passing from one stage to
the next within half a day. The sensitivity of units to visual stimuli increased
rapidly during this period of development. By stages 45 and 46, the majority
of the units we have examined (20 out of 23) gave strong 'on' responses to
ambient illumination. Not only were the responses more vigorous but they were
more resilient to repeated stimulation than those observed at stage 43.
A notable phenomenon during the early stages of development was the rapid
924
S. H. CHUNG AND OTHERS
emergence of 'off' components in the visual responses. Although the units in
stages 45-46 gave predominantly 'on' responses to ambient illumination, 'off'
responses consisting of two to three spikes could be elicited by using a bright
background illumination. Units also began to respond to a black stimulus
traversing the receptive field, provided the size of the stimulus exceeded 15°
and had a high contrast against the background. The size of the receptive field
thus delineated generally subtended 30-40° of visual angle.
By stage 47 units generally responded to a 2° spot of light. With this, the
relative strengths of the 'on' and 'off' responses were plotted at various points
across the receptive field and expressed in terms of visual angle (Fig. 5). After
the centre of the receptive field was located the spot of light was turned on and
off at a fixed rate (on for 2 sec and off for 3 sec) and the number of impulses
at on and off was counted. The spot was then moved across the receptive field
in steps of 5° of visual angle. When the receptive field was mapped in this
manner its shape tended to be circular or sometimes oval, the size ranging from
30° to 40° of visual angle. As can be seen in Fig. 5, the relative strengths of 'on'
and 'off' responses changed with advancing stages of development and the size
of receptive field decreased gradually.
Up to stage 48 the units we have encountered most frequently (40 out of 42)
were 'on-off' type with simple receptive field organization. They responded
briskly to a large black disc traversing the field, although the responses tended
to habituate quickly to repeated stimulation. Equally strong, if not stronger,
responses were obtained to changes in ambient illumination as to a small spot
of light in the receptive field. These properties of the transient stimulus-response
are similar to those of adult 'event' units (Lettvin, Maturana, McCulloch &
Pitts, 1959). In the absence of any systematic stimulation units were either totally
silent or discharged sporadically, the rate of discharge ranging from 2 to 5
spikes per min in the dark.
Stages 49-52. At around stage 49 a new type of unit appeared. These were
characterized by their acute sensitivity to small changes in the total flux of
illumination. The units exhibited the property of adult 'dimming' units (Lettvin
et al 1959) in that a burst of spikes was elicited by each step wise darkening of
the background light. However, unlike 'dimming' units in adult Xenopus, these
units responded equally well to step wise brightening of ambient illumination
(Fig. 6). This latter component became less obvious with advancing stages, and
finally by stage 56 the majority of the units ceased to respond to brightening.
One of the salient features of early 'dimming' units was their vigorous endogenous activity. The presence of this background discharge was apparent in
most animals by stage 49, although in some animals its onset was delayed by
one or two stages. The rate of discharge increased rapidly, reaching its peak
around stage 51, and subsided to a very low rate thereafter. Units discharged,
at the height of their hyperactivity, with more than 500 spikes/min, about four
times the rate found in adult 'dimming' units.
Development of retina in Xenopus
925
1 sec
Fig. 6. Response of an early 'dimming' unit in a stage-49 tadpole. The unit discharged in response to both a gradual increase (upper record) and decrease (lower
record) of ambient illumination. The line below the spike trace indicates the relative
level of illumination, which was increased and then decreased by 3 log units.
At stages 49-50, 'dimming' units discharged equally well in the light as in the
dark (Fig. 7). The upper record in Fig. 7 is a spectrum of interspike intervals
in the dark; when the background illumination was introduced (lower record
of Fig. 7) there was a brief inhibition. The unit then resumed its endogenous
discharge at approximately the same rate as it had done in the dark. However,
by stage 53 the rate of endogenous activity was markedly suppressed when the
retina was illuminated and this activity took longer to reappear.
Stages 53-60. Of the 72 units we examined in tadpoles from stages 53-60,
35 % could be classified as 'event' units and 37 % as 'dimming' units, with the
remaining showing characteristics of a mixture of the two types. Such a unit
would, for instance, respond to the first three steps of stepwise dimming as
well as to a spot of light on and off.
The size of the receptive field decreased markedly during these stages of
development. The receptive field, when measured with a 2° spot of light,
generally subtended 30-40° of visual angle at stage 53 and its size steadily
decreased to 10-15° by stage 57.
Stages 61-66. The first sign of sustained responses were seen occasionally
around stage 61 and could reliably be obtained in metamorphic tadpoles in the
mid-visual field. The best responses were obtained by a black disc subtending
10° of visual angle. The size of the optimum stimulus closely corresponded to
the size of the receptive field delineated with a spot of light. Unlike those in
adults, 'sustained' units in tadpoles fatigued quickly. To the first presentation of
a 10° disc in the receptive field, units responded with a sustained discharge. On
926
S. H. CHUNG AND OTHERS
1U
A
i
103
102
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10
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... ,.f,........ .... ....,..... .
1
104 •
I
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50
100
B
1
103 •
, •
10 2 •"
•
,
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, . " . ' ' '
• '
.• : ;. l >-:i...-;v-.»V«-.;5!'.?
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Time (sec)
100
Fig. 7. The CLOOGE records of endogenous activities of a 'dimming' unit in a
stage-50 tadpole. Each dot denotes the occurrence of an impulse, and its vertical
displacement gives the duration of the interval between the impulse and the preceding one.
(A) Endogenous activity of the unit under ambient illumination is shown in the
first segment of the record. At downward arrow, ambient light was turned off. The
'off' discharge, lasting approximately 10 sec, was followed by the resumption of
the endogenous activity.
(B) Endogenous activity in the dark was briefly suppressed when ambient illumination was reintroduced (downward arrow). The rate of discharge returned to the
same level as that in the dark.
the second and subsequent presentations, responses did not sustain and became
very similar to those of an 'event' unit (Fig. 8). Nevertheless, these units were
distinguishable from 'event' units, as they were generally unresponsive to on
and off of ambient illumination.
The layering of the incoming optic fibres according to unit type became
evident during this period of development. 'Dimming' units were always
Development of retina in Xenopus
I I Mil I II 1 I I Mllll
A TT7
I I (
II II!
Ml 1 Ml II I I
927
I
'ft
llff\
/ / r r r r / rri i i i i r/ i n ITTTT r i T T r n T T r r
V.
1 sec
Fig. 8. Response of an early 'sustained' unit in a stage-64 tadpole. The unit gave a
sustained discharge when a 15° black disc was moved in its receptive field and held
there stationary (upper record). Introduction of the stimulus in the receptive field
is denoted by the downward excursion of the line below the spike trace. To the
second and subsequent presentation of the stimulus, the unit responded with brief
bursts of impulses and did not show a sustained response.
found deeper down in the tectum than 'event' units, with the rudimentary
'sustained' units lying close to the surface of the tectum.
Endogenous discharge patterns of different unit types showed a large variability, both from one animal to another and between different units in an
animal. 'Sustained' units remained silent either in the dark or in the light. 'Event'
and 'dimming' units, on the other hand, discharged sporadically, but there was
no obvious patterning of discharge such as that characteristic of adult animals.
Adult (1-2 years old). The organization of different types of responses recorded
from the optic nerve terminals in adult Xenopus closely resembled the original
observations on Rana pipiens made by Lettvin et al. (1959). Unmyelinated optic
fibres terminated in the superficial layer of the tectum, while myelinated fibres
comprising 'event' and 'dimming' fibres extended their terminals beneath this
layer (Chung, Bliss & Keating, 1974a).
Transient response characteristics of'event' and 'dimming' units were identical
to those found in metamorphic tadpoles. 'Sustained' units, however, underwent
considerable changes during the post-metamorphic period. The optimum size
of the stimulus for these units was a 2° black disc in the receptive field. Unlike
those in the metamorphic tadpoles, 'sustained' units in adult showed little, if
any, sign of fatigue to repeated presentation of the stimulus.
There were two notable changes in unit activity between metamorphosis and
young adults. First, each class of units exhibited a particular pattern of endogenous discharge. Because sequential pulse intervals varied widely, clusters of
long and short intervals occurred, giving a rhythmic 'style' to the time series.
The internal organization of the firing pattern was sufficiently distinct for the
experimenter to be able to define the unit type from the nature of this endogenous activity. The pattern could best be preserved by plotting successive
interspike intervals against time as shown in Fig. 9. Endogenous activities of
928
S. H. C H U N G AND OTHERS
Sustained unit
Event unit
10s
104
Dimming unit
103
102
102
-
'
'
.
•
•
•
:
•
•
.
• *-.J»ti.'i).*'(•**•' ;fc'it,» •«»'• H.'l
£ I 10°
10°
10°
c
I
10
20
1
10
20
Time (min)
1
1
10
20
Fig. 9. The CLOOGE records showing the characteristic endogenous discharge
patterns of 'sustained', 'event' and 'dimming' units in adult Xenopus. The records
were obtained from three different units discharging in the dark after the retina was
fully dark-adapted.
'sustained' and 'event' units exhibited essentially the same patterns both in the
dark and under the ambient illumination. The activity of 'dimming' units, on
the other hand, was influenced both by the level of illumination and the state
of retinal adaptation, as described previously (Chung, Raymond & Lettvin,
1970).
Secondly, the receptive field properties of the three unit types showed more
complex organization than those found in pre-metamorphic animals. The complexity entailed a pronounced interaction between the receptive field, delineated
by a black disc, and the peripheral visual field surrounding it. The existence of
a complex receptive field with inhibitory surround in 'sustained' units could be
revealed by various methods. Large discs evoked less activity than small ones
when moved through the receptive field (cf. Maturana, Lettvin, McCulloch &
Pitts, 1960; Butenandt & Griisser, 1968). Similarly, when two discs were moved
across the visual field, one traversing the receptive field and the other passing
outside it, the resultant response was less vigorous than in the absence of the
outside disc. Keating & Gaze (1970) showed that a sustained discharge evoked
by a 3° disc in Rana could be inhibited by a movement of a large object in the
vicinity of the receptive field. Using this technique, we could demonstrate that
the activity of a unit could be affected over a wide visual angle, as illustrated in
Fig. 10. The upper record shows the sustained response to a 3° black disc
moved into the receptive field and held stationary. The lower record shows the
effect of peripheral stimulation on the sustained activity of the unit; the sustained response was suppressed by the introduction of a large disc 45° away
from the receptive field.
Peripheral inhibition of 'event' units could be revealed in a similar way. The
endogenous discharge of these units was extremely susceptible to stimulation in
other parts of the visual field. When a portion of the visual field some distance
away from the receptive field was stimulated, the endogenous activity was partially, or sometimes completely, suppressed. In Fig. 11, the endogenous activity
of an 'event' unit under ambient illumination is plotted against time. At the
Development of retina in Xenopus
929
•
*
1 sec
Fig. 10. Peripheral inhibition of a 'sustained' unit in adult Xenopus.
(A) A sustained response to a 3° disc moved in the receptivefieldand held there, as
indicated by the bar below the spike trace.
(B) The sustained response to the 3° disc was inhibited when a 25° disc was introduced (wide bar) in the visual field 45° away from the receptive field. The unit
resumed its activity when the inhibiting stimulus was removed.
1
1U
\
103
I*'
102 -
* •.
«
10 -
40
80
1
1
120
200
Time (sec)
Fig. 11. The CLOOGE record showing peripheral inhibition of endogenous discharge of an adult 'event' unit. The endogenous activity of the unit under ambient
illumination was suppressed totally when, at the first arrow, the visualfield80° away
from the receptivefieldwas stimulated with a 25° disc. The activity resumed when the
inhibiting stimulus was removed (second arrow).
930
S. H. CHUNG AND OTHERS
104
\
103
102
' !
10
20
40
60
104 *
103
•
•
* 1
•ft*. •
102
10
•
*
20 •
40-
60
Time (sec)
Fig. 12. The CLOOGE records showing peripheral interaction of a' dimming' unit in
adult Xenopus.
(A) The typical responses of a 'dimming' unit to on and off of ambient illumination
in the receptivefield.The endogenous activity is suppressed when ambient illumination is introduced (first arrow), and 'off' discharge is elicited upon the cessation of
the illumination (second arrow).
(B) When a strip of the visual field, not encompassing the receptive field and 30°
away from it, was stimulated, the unit now responded to light on and was inhibited
to light off.
first arrow, a 25° disc was moved repeatedly some 80° away from the receptive
field. The activity ceased almost completely until the inhibiting stimulus was
removed at the second arrow. A brief burst of impulses, reminiscent of the
phenomenon of disinhibition, was followed by the resumption of the endogenous discharge.
As in the cases of 'sustained' and 'event' units, all 'dimming' units also showed
complex field organization. When stimulated with a 2° spot of light, 'on' responses
Development of retina in Xenopus
931
could be evoked from certain isolated regions of the visual field up to 90°
away from the main receptive field (cf. Keating & Gaze, 1970). This peripheral
interaction could also be demonstrated by moving the perimeter arc so that it
did not include the receptive field proper of the unit, whereupon the unit
abruptly changed its operating characteristics and discharged at light on instead
of off (Fig. 12).
DISCUSSION
Correlation between the anatomy and physiology of the retina
Within a relatively brief period of time, the retina of Xenopus undergoes a
structural transformation, from a mass of undifferentiated cells to a stratified
structure. We have described these changes and related them to concomitant
changes in the receptive field properties of retinal ganglion cells.
By the end of the embryonic period, ganglion cells spread out into a single
layer, and the inner and outer plexiform layers become separated by a stratum
formed by the inner nuclear layer (Fig. 1). The first sign of visually evoked
electrical responses appears at this stage, consisting predominantly of 'on'
discharge to bright illumination. The absence of an 'off' component in the early
responses suggests that the neuronal network for evoking 'off' responses requires further maturation.
Between stages 44 and 51, there occurs a marked increase in the thickness
of the inner plexiform layer (Fig. 2). This process is accompanied by an increase
in the sensitivity of visual units and the emergence of 'off' responses. Two classes
of visual units become clearly discernible. Maturana et al. (1960) postulated
that different classes of units are associated with different shapes of ganglion
cell dendritic trees. Thus, for instance, the 'dimming' responses may be elicited
from ganglion cells with T-type dendritic fields, whereas those with H-type may
give rise to 'event' responses. The present observations do not enable us to make
any useful comments on this tentative functional classification of ganglion cells.
Adequate proof of such an identification requires an intracellular staining technique, which we have not employed for the present study. However, our Golgi
impregnation of retinal ganglion cells reveals that there are only two types of
retinal ganglion cells, the T-type and H-type (Fig. 4D & E), in the mid-larval
tadpoles. Physiologically, two classes of units are identifiable in these animals.
Just before metamorphic climax, a new type of retinal ganglion cell appears
in the histological sections (Fig. 41 & J). The timing of this coincides with the
appearance of 'sustained' units in visual responses (Fig. 8). This observation
enables us to make a tentative functional classification of the constricted-tree
retinal ganglion cells (cf. Pomeranz & Chung, 1970). The reason for the late
emergence of 'sustained' units is not known; it is highly unlikely that these cells
develop by mitosis late in larval life. The ganglion cells comprising central
retina are still the original ones that first developed in this position (Straznicky
& Gaze, 1971). Hollyfield (1971) has stated that extensive mitosis takes place in
58
EMB 33
932
S. H. CHUNG AND OTHERS
the inner nuclear layer late in tadpole life. What proportion of these lateappearing cells in the inner nuclear layer are neurons is unknown; the evidence
presented (Hollyfield, 1971) indicates that some of them are glial elements. It
seems most probable that 'sustained' units (ganglion cells) slowly evolve from
pre-existing 'event-type' units by the formation of additional synaptic networks.
A pointer in this direction is the fact that the distinction between 'event' and
'sustained' units is not as conspicuous in tadpoles as it is in adult life (Fig. 8).
Also, the very extensive increase both in the thickness of the inner plexiform
layer and in certain classes of synapses in the layer (Fisher, 1972) in later tadpole
stages, suggest that this particular integrative mechanism is altering markedly.
Depth distribution of optic affevents in the tectum
Throughout larval life in Xenopus, 'event' and 'dimming' units arborize in
the superficial layer of the tectum, forming excitable synaptic contacts with,
presumably, apical dendrites of tectal cells (Chung, Keating & Bliss, 19746).
These optic nerve fibres are mostly unmyelinated in young animals and the
number of myelinated fibres increases steadily with maturation (Gaze &
Peters, 1961; Wilson, 1971). In adult animals, different types of ganglion cells
distribute their axon terminals in different layers of the tectum. Myelinated
'event' fibres, for instance, synapse on the shaft of dendrites just above layer 8
of P. Ramon (1890), whereas the synaptic sites for 'dimming' fibres are located
below layer 8, forming synaptic contacts perisomatically and probably also with
the basal dendrites of tectal cells in this layer (Chung et ah 1974 a).
These results suggest that the sites of synapses in tadpoles differ from those
in adults. If this is so, two plausible explanations could be proposed. First,
unmyelinated nerve fibres giving rise to 'event' and 'dimming' responses in
tadpoles may remain unmyelinated, and their receptive field properties change
into 'sustained' types with development. Although we do not have a direct
evidence against this possibility, we think it highly unlikely. 'Event' and 'dimming' units, as noted previously, appear before stage 50, and subsequent development of these units can be seen to result in further refinement of receptive field
characteristics. Two branching patterns of the dendritic tree, which are believed
to be those giving rise to the two response classes (Lettvin, Maturana, Pitts &
McCulloch, 1961), are identifiable histologically. The second, and more
plausible, explanation could be that synaptic contacts formed during embryonic
development are transitory and shift continually during the course of retinal and
tectal maturation. Thus, optic nerve fibres which arborize initially in the superficial layer of the tectum, forming transitory synaptic contacts with the apical
dendrites of tectal cells, may gradually shift to a deeper layer of the tectum
during the course of tectal maturation.
Such a flexibility in synaptic relationships has been adduced previously.
During the development of the retino-tectal projection, a given optic nerve
fibre arborizes consecutively at a series of tectal sites (Gaze, Chung & Keating,
Development of retina in Xenopus
933
1972; Gaze et al. 1974; Chung et al. 19746). Our present findings, taken in
conjunction with previous studies, lead us to postulate that synaptic contacts
between optic nerve fibres and tectal cells are labile during development and
that the adult patterns of retinotopic and stratified organization obtain only as
the end result of a process during which many temporary synaptic relationships
are made and broken.
Peripheral inhibition and endogenous discharge
The properties of the receptive fields of ganglion cells do not cease to change
with metamorphosis. The transient response characteristics of visual units
(except 'sustained' units) in recently metamorphosed Xenopus are similar to those
in adults, but two salient features of adult visual units, namely the endogenous
discharge and the peripheral inhibition, are notably underdeveloped and only
appear sometime after metamorphosis. If the visual system develops further
with maturation, the changes we observe after metamorphosis must be important features of its function.
It has been shown in this paper that each neuron type in adult animals has
its distinct pattern or 'style' of firing (Fig. 9), and the pattern of this background
discharge can undergo subtle alteration as a response to visual events (cf.
Chung et al. 1970). Because style becomes a meaningful notion, it is necessary
to ask how it is generated. Broadly speaking, three factors must be involved;
first, the nature of the synaptic inputs to the neurons; secondly, the way these
inputs are ordered on the dendritic tree; and finally-a point well emphasized
by Lettvin et al. (1961) - the geometry of dendritic ramification. All these
factors are known to change during maturation. The thickness of the plexiform
layers, as well as the receptor layers, increases throughout larval and adult life
(Fig. 2). Fisher (1972) noted that the number of synapses in the inner plexiform
layer, especially conventional and serial-conventional types, increases markedly
in the later tadpole stages. The geometry of the dendritic trees of the ganglion
cells undergoes similar changes during development (Fig. 4). These factors, all
considered, lead us to expect that the styles of firing will undergo gradual
changes as the animals mature. In so far as different classes of neurons have
their distinct shapes of dendritic tree, thereby receiving different sets of inputs,
the styles of discharges too will differ from each other.
The size of the receptive field of a ganglion cell, when measured with a small
spot of light or black disc, remains approximately constant after the mid-larval
stage. However, a receptive field is surrounded by a zone of inhibitory area,
which is absent in developing tadpoles and expands progressively with maturation. This zone of inhibitory periphery appears to expand to include the entire
expanse of the retina in adult Xenopus of 2 years old (Figs. 10-12). Previous
estimates of the extent of this inhibitory region in Rana varied. Schipperheyn
(1965), for instance, claimed that the inhibitory region of Class II neurons
58-2
934
S. H. CHUNG AND OTHERS
extended over a distance of only about four receptor cells, or 0-34°, whereas
the estimate of Pickering & Varju (1967) was 5° or more. Subsequently, it was
demonstrated that the activity of these units could be inhibited by exciting a
part of visual field which was 30° away from the unit's excitatory receptive
field (Keating & Gaze, 1970).
We maintain that the outer limit of a receptive field is dependent on the technique one uses to test it. We have demonstrated that the endogenous discharge
of a neuron can be suppressed partially or completely by exciting part of the
visual field which is 80° away from the receptive field (Fig. 11). These results are
hardly surprising, since we are not dealing with a single ganglion cell lying in a
volume conductor. In a dense neuropil, where dendritic trees and presynaptic
elements are interdigitated in a massive tangle, the external current flow of any
one element must pass through its neighbours as part of its external space.
Whether such an interaction is mediated by passive spread of the external current through the S-space, as Naka (1972) postulates, remains to be shown. The
fact that such peripheral inhibition is absent in developing visual units at early
stages and appears and becomes more marked with advancing age suggests
that the mechanism mediating this lateral interaction develops progressively
with maturation.
Appendix:
Optics of Xenopus eyes during development
By M.
L A N D AND R.
VICTORIA
STIRLING
Xenopus are wholly aquatic as larvae, but amphibious as adults, spending a
considerable part of the time with their eyes, at least, above the surface. The
questions thus arise, are Xenopus eyes optically suited for use in air or water,
and is there an optical change at metamorphosis? According to Walls (1967)
adult frogs and toads are optically adapted to air, with the cornea acting as an
important refracting surface, and have insufficient accommodation to focus an
image on the retina in water when the cornea is optically ineffective. In the
tadpole, however, this may not be so; the lens is spherical, as in fish eyes and
unlike the rather flattened lenses of adults, and it is at least possible that the
image it forms lies on the retina. In this Appendix however, we establish that
the eyes of both larval and adult Xenopus are somewhat hypermetropic in water,
in spite of the fact that the larval lens is similar in optical properties to that of
fish.
Focal length of the lens. Fish lenses have a refractive index that decreases from
the centre to the periphery, where it is little greater than that of water. This
gradient produces two desirable properties (see Pumphrey, 1961). First, because
light rays are bent continuously in the lens, and not just at the interfaces, the
focal length is reduced from about four radii (which it would be if the lens had
Development of retina in Xenopus
935
F=2-5R
mm
Stage 46
8 cm adult
05
Lens radius (mm)
Fig. 13. (A) Drawing of the eye of a Xenopus larva at about stage 57, based on
measurements described in text, showing the image-forming cone and the positions
of the rod layer (r) and the image (i) relative to the back of the pigment layer. The
lines beneath indicate the lens diameters of a stage-46 larva and an adult. (B) Focal
lengths of 17 Xenopus lenses as a function of their radii.
a homogeneous refractive index equal to that at its centre-about 1-51) to
approximately 2-5 radii: this value is constant for virtually all fish and is known
as Matthiessen's ratio. Secondly, in a homogeneous spherical lens rays passing
through the outermost zones are brought to a focus much nearer the lens than
the focal point for rays close to the axis, resulting in very severe spherical
aberration. In a fish lens this is eliminated by the fact that peripheral rays are
bent relatively less by the low refractive index of the outermost zones. Fletcher,
Murphy & Young (1954) have shown that for this aplanatic condition to be met,
there is a unique refractive index gradient for a given focal length, and they
calculate this gradient. Fig. 13 shows that for all larval stages, Xenopus lenses
conform quite exactly to Matthiessen's ratio, and it can be assumed from this
that their construction is essentially similar to that of fish lenses.
Lenses were removed from the eyes of nine animals immediately after they were killed, and
were suspended in a flat-bottomed glass dish containing frog Ringer. They ranged in diameter
from 300 /<m (stage-46 larva) to 2100 /*m (adult: 8 cm body length). Their focal lengths were
determined by measuring, with the fine focus of the microscope, the distance from the lens
centre (focusing on the visible lens periphery) to the circle of least confusion produced by a
point source at a large (30 cm) distance in front of the lens. The measurement was made in
Ringer but with the microscope in air, and thus the real distance is the measured distance
multiplied by 1 -333, which is taken to be the refractive index of Ringer. The accuracy of this
method is estimated to be better than 20 /tm for the smallest and 50 /*m for the largest lenses.
Lens radii were measured simultaneously. The objective used had a n.a. of 0-3, which meant
936
S. H. CHUNG AND OTHERS
• Centre of lens to retina (mm)
1
a
Retina
£ 2
(L)
j2
£<
§
Back of
pigment layer
\
^"°
u
Fig. 14. Image position as a function of the axial length of the eye from lens centre
to the rod layer of the retina. • , Measured image position with the cornea in
water. O, Calculated image position for adults with the cornea in air.
that it was accepting a 26 ° cone of light from the lens; the whole cone of image-forming light
from the lens occupies approximately 44 °. In one case this estimate of the focal length was
checked by measuring the image magnification of a grid at a known distance from the lens:
the value obtained was the same as that obtained by image distance measurement, to within
2°/
*•
/<>•
With the exception of one or two lenses which had some adhering tissue, all
lenses gave excellent point images of point sources, and well-resolved images of
grid patterns. The image quality was much better than it would have been,
using an n.a. 0-3 objective, if the lens did not approach the aplanatic condition
closely. Certainly up to metamorphosis these lenses are likely to contain a
refractive index gradient closely similar to that given by Fletcher et al. (1954).
This raises the interesting problem of how a lens manages to retain the same
gradient, with respect to its increasing radius, throughout the 3-fold increase in
linear dimensions that occurs between stage 46 and metamorphosis. The six
adult lenses studied were slightly flattened (the ratios of axial to equatorial
diameters lay between 1:1-05 and 1:1-08 - much less than the figure of 1:1-13
given by Walls (1967) for adult frogs), but the focal length:equatorial radius
Development of retina in Xenopus
937
ratio, even in the oldest Xenopus, was only 2-60, indicating that there is no
substantial departure from the 'fish design' after metamorphosis (Fig. 13 B).
Position of the image. The position of the image, in water, can be found by
determining the distance from the centre of the lens to the centre of the rod
layer of the retina, and comparing this distance with the focal length of the
excised lens. The results, for the same 17 eyes as were used for Fig. 13, are
shown in Fig. 14.
Eyes were either photographed or drawn with a camera lucida after they had been aligned
so that their optical axes were horizontal. This was done in larvae by anaesthetizing them,
tilting them slightly, and measuring the dimensions in situ. In adults, eyes were carefully removed immediately after death, aligned, and photographed in Ringer. The eyeball did not
deflate within the first 5 min after removal from the orbit. The distance from the front of the
lens to the back of the pigment layer was measured directly. From this was subtracted the
radius of the lens, determined after excision, and the distance from the back of the pigment
layer to the centre of the rod layer, measured from unfixed histological preparations. The
combined errors are estimated to be of the same order as those of the focal length measurements - 20 /<m for the smallest and 50 /tm for the largest eyes. The two methods of measurement were checked against each other by measuring the same metal object from the side
using an eye-piece graticule, and by rotating it and measuring its depth in water using the
fine focus: the results agreed to within 1 %.
Fig. 14 shows that in water the focal point for all eyes lay behind the retina,
and in all but four eyes behind the pigment epithelium. The distance between
focal point and retina increases approximately with the size of the eye, although
variability and measurement errors make it impossible to say whether the
relation is exactly linear. Expressed as a percentage of the focal length, the
average mismatch between image and retina is 14-5 % for the eleven larval eyes
and 17-0 % for the six adult eyes. Since these distances are in the hypermetropic
direction, objects close to the eye will increase these mismatches, so that there
is no point in front of the eye which is conjugate with the retina in water.
In air the situation is quite different because the cornea becomes a refracting
surface. The position of the image formed by the cornea is first determined
from the radius of curvature of the cornea (r):
71—1
where n is the refractive index of water (1-333) and d is the distance from the
cornea to the image. Then the final image position (d) is found by taking the
corneal image as the virtual object for the lens, which can be treated as a thin
lens because its principal and nodal points coincide with its centre:
f+(d-s)>
where/is the focal length of the lens, s the axial separation of cornea and lens
centre and d' the required distance from lens centre to image. The image position
for the six adult eyes is shown by open circles in Fig. 14. For the four smaller
eyes the image in air lies about as far in front of the retina as the image in water
938
S. H. CHUNG AND OTHERS
lies behind it; for the two eyes of the largest animal the image lies almost
exactly on the retina in air, for objects at infinity. For objects closer to the eyes
the image will approach the retina of the smaller adults. For the six adult eyes
the image in air lies an average of 9-3 % of the focal length of the lens in front
of the retina.
Optical consequences of a large defect of focus
If the retina and image plane do not coincide, as seems to be the case for both
larval and adult Xenopus in water, then the image of a distant point source will
be a blur circle on the retina. The diameter of the blur circle (b) is given by
f '
where a is the pupil diameter which is typically about three-quarters of the lens
diameter, and df/fis the distance from the image to the retina divided by the
focal length of the lens. As previously given, this is 0-145 for larvae and 0-170
for adults. The size of the blur circle can be expressed as a visual angle (angle
subtended at the nodal point of the eye which is the centre of the lens) which is
then independent of the size of the eye. This is given by
o
==
, ~ ~ T^\ radians,
°Jj)
or (57-3 x b)l(8f-f) degrees, where (Sf—f) is the distance from the centre of the
lens to the retina, which is equal to the focal length minus the distance from the
image to the retina. For larvae the average diameter of the blur circle on the
retina should be approximately 6-3° and for adults (in water) 7-3°. In air the
blur circle should be slightly smaller than this for both larvae and adults,
possibly decreasing to nearly zero for fully mature animals.
It is difficult to make predictions about the ability of such a system to resolve
dark or light spots or lines, because the performance of the retina is then
essentially limited by the ability of ganglion cells to detect small temporal
changes or spatial differences in intensity. All that can be said is that one would
expect responses to decline when the size of the stimulating spot is smaller than
the angular size of the blur circle. However, the situation is different for gratings,
and it is simple to show that for extended square-wave gratings the contrast on
the retina should fall to zero when the spatial period of the grating is equal to
the diameter of the blur circle (this is only true if the defect of focus is large and
explicable by geometrical optics, and it is here). One would thus expect that
Xenopus larvae in water would not give optomotor responses to striped drums
whose spatial period was less than 6-3°, although for adults in air the spatial
period should be very much less than this, if only optical considerations are
involved.
Development of retina in Xenopus
939
One small point that should be made is that towards the far nasal and temporal edges of the retina the pupil cuts off light from most of the lens. Thus the
size of the blur circle here is much reduced, to 2° or less in the larvae, and
resolution might be enhanced, although the brightness of the image will be
substantially reduced, for the same reason.
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