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The projection of optic nerve fibers in the frog Rana catesbeiana as studied by radioautography.

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The Projection of Optic Nerve Fibers in the Frog Rana
catesbeiana as Studied by Radioautography
STEPHEN GOLDBERG AND MINORU KOTANI
Departments of A n a t o m y a n d Pathology, Albert Einstein College
of Medicine, N e w York, N . Y.
ABSTRACT
Tritiated leucine was unilaterally introduced into the vitreous chambers of metamorphosing bullfrog tadpoles in a n attempt to determine whcther the
optic fibers may be traced by radioautography. After injection, the animals were sacrificed at varying time intervals from 4v2 minutes to 32 days. Serial sections of the
brain were subjected to radioautography. The optic fibers were thereby traced to their
known terminations and to a small thalamic cell cluster to which we found no reference in the literature. Significant differential radioactivity was detectable i n the contralateral optic lobe as early as six hours postinjection, suggesting a flow, whether
intra- or extraaxonal, of at least 10-22 mm/day.
A single postmetamorphic frog underwent excision of the optic chiasin with positioning of the stumps of the optic nerves against the lateral wall of the thalamus.
Fifteen weeks were allowed for regeneration. When the same histologic and radioautographic technique as above was used (sacrificing at eight days postinjection), a
grain path was found to extend from the optic nerve into the brain. This path divided
into two distinct branches, one branch crossing over and one remaining on the same
side. These results indicate the practicality of tracing nerve distribution within the
CNS by radioautography.
The concept of axonal protoplasmic flow
has in recent years been studied by means
of radioautography (Droz, '65; Droz and
Leblond, '63; Turbes, '65; Weiss, '65). It
has been shown, for instance, that, following the systemic administration of labeled
amino acids i n the rat, radioactive tracings
can be detected, at first, in the perikarya
in various central nervous system areas
and then, after several days, in their corresponding axons (Droz and Leblond, '63).
Other investigators have reported on the
rate of movement of labeled material along
the optic nerve following local injections
of tritiated amino acids into the vitreous
chamber (Turbes, '65; Weiss, '65).
These experiments have given rise to
the suggestion that radioautography could
be used as a technique for tracing neural
pathways. In theory, if axonal flow is a
universal phenomenon, one should be able
to follow the axonal projection from a
given region by making focal injections of
labeled amino acid into that area, sacrificing the animals at selected time intervals, and then examining serial sections
of the specimens after a suitable period of
radioautographic exposure. If such a technique could be perfected and applied to
ANAT. REC., 158: 325-332.
nuclei within the CNS, one could possibly
circumvent one of the difficulties of classical methods, which employ focal lesions
to produce degenerative pathways. A lesion
in a cluster of cells destroys, in addition
to the cell bodies of the cluster itself, the
axons which pass through the cluster from
other regions. Hence, one sometimes does
not know whether the degenerative pathway originated in the perikarya of the lesion site or elsewhere. If, however, labeled
amino acids are selectively absorbed by
perikarya rather than axons, then the injection should result in the labeling only
of the pathways originating in the cell
bodies of the injection site. Whereas there
is recent evidence that amino acid can be
directly incorporated into axons (Singer
and Saltpeter, ' 6 6 ) , the amount would appear to be insignificant compared with the
amount absorbed by perikarya (Droz and
Leblond, '63).
The object of our experiment was to use
radioautography to trace out the connections of a relatively simple neural pathway. The frog optic nerve was selected for
study. Since it decussates virtually completely at the chiasm, a comparison of the
two sides, it was felt, should enable one to
325
326
STEPHEN GOLDBERG AND MINORU KOTANI
distinguish label brought by the blood
(general label) or by other fluid movement,
from that due to axonal flow or other fiberlimited conduction. Metamorphosing tadpoles were used because it was thought
that a growing animal would have a faster
flow than an adult.
jected with 10 yc of tritiated leucine. Eight
days postinjection, the animal was sacrificed and submitted to the histological
and radioautographic procedures described
above.
RESULTS
A. Metamorphosing tadpoles.
In the
specimen
sacrificed
after
four
and
one-half
MATERIALS AND METHODS
minutes, 20 1.1 sections showed heavy grain
A. Metamorphosing tadpoles. A series deposits over all layers of the right retina,
of 13 metamorphosing Rana catesbeiana extending through the eyeball in a de(bullfrog) tadpoles, approximate weight creasing gradient to the ipsilateral surface
15 g each, were placed under light anaes- of the brain. More grains were found in
thesia (tricaine methane-sulfonate); 5- the ipsilateral half of the brain than con25 pc of tritiated leucine (DL-leucine-4,5- tralaterally. Within the retina, there apH3 hydrochloride (spec. activity 5.5 curies/ peared to be a somewhat heavier grain
mmole; 1 mc/mlj were injected into the concentration over axonal layers than over
right vitreous chamber of each tadpole. cell bodies.
The animals, kept in plastic containers in
At six hours (20 p sections), the greatwater a t about 23"C, were sacrificed a t est concentration of grains again was withfour and one-half minutes, six hours, and in the retina and again there appeared to
1, 2 , 4, 5, 6, 8, 12, 15, 18, 22 and 32 days, be more grains over axonal regions than
and fixed in Bouin's solution for 1-3 days. over cell bodies. The concentration of
(Specimens sacrificed at four and five grains diminished as the optic nerve was
days were unsatisfactory for technical rea- traced toward the brain, as in the four and
sons). The brain, attached to optic nerves one-half minute specimen; unlike this
and retinae, was dissected out of the cra- specimen, though, the contralateral marnium, in 70% alcohol. After histological ginal optic tract, as f a r as its termination
processing, the slides were deparafin- in the contralateral optic lobe, displayed
ized, horizontal and cross-sections (5- a slight but significant increase in grain
40 1.1, mostly 20 u j were dipped in Kodak concentration over the corresponding ipsiNT-3 emulsion, exposed for 24 days to lateral areas. These observations were confive and one-half months, and developed firmed on specimens sacrificed after one
with D-19 diluted 1 : 1 with distilled water. and two days (5 1.1 and 10 u, respectively).
Slides were stained with H and E. ReThe remaining animals each showed a n
sults were recorded by projection and pho- uninterrupted pathway of grains extendtography.
ing from the retina to the optic nerve,
As controls for background label, non- across the chiasm, to the contralateral
injected animals were submitted to radio- marginal optic tract, terminating at the
autography for a comparable period of first cellular layer of the contralateral optime. I n addition, as a control against pig- tic lobe (figs. 1, 2 ) . This agrees with the
ment granules, which are prominent in known distribution of the fibers as studied
frog brains, a series of slides not submitted by traditional methods.
In addition, three grain paths were
to radioautography was prepared.
B. Postmetamorphic frog - regenerat- noted to separate from the contralateral
ing optic nerve. As the frog optic nerve marginal optic tract at separate points :
is capable of regenerating, a single at( 1 ) A basal grain pathway was obtempt was made to trace out the course served to extend ventral to the marginal
of a transplanted regenerating optic nerve. optic tract to a marginal point near the
After the manner of Sperry ('45 j , the optic border between thalamus and hypothalachiasm was removed in a young post- mus where it turned medially, and dismetamorphic bullfrog and the optic nerves appeared into a small cluster of cells
pushed dorsally to grow into a more dorsal corresponding to the nucleus lateralis tegregion of the thalamus. Fifteen weeks menti of Herrick (Herrick, '25; Kappers
later, the right vitreous chamber was in- et al., '60 j . This nucleus is a known termi-
Fig. 1 Drawings of cross sections through the tadpole brain at the optic chiasm ( A ) , thalamus
in the region of the first thalamic branch ( B ) , and anterior region of the optic lobe ( C ) . Shaded
areas indicate regions of heavy silver grain concentrztions following injection of the eye with tritiated leucine, indicating the course of the optic nerve.
328
STEPHEN GOLDBERG AND MINORU KOTANI
Fig. 2 Photographs of sections through the optic chiasm in tadpoles with right vitreous
chambers injected with tritiated leucine. ( A ) Cross section; sacrificed 15 days postinjection,
X 90. (B) Horizontal section; sacrificed six days postinjection, x 130. Arrows indicate direction of optic nerve projection which is seen as a deposition of silver granules; r.o.n.,
right optic nerve; m.o.t., marginal optic tract; o.c., optic chiasm.
NERVE TRACING VIA RADIOAUTOGRAPHY
329
nal connection of optic nerve fibers in the way for the background adaptation response of the pars intermedia.
frog (fig. 1 C ) .
B. Postmetamorphic frog - regenera( 2 ) A second grain path separated from
the marginal optic tract to curve dorsally tion of optic nerve. Throughout the 15
around the tractus striathalamicus et hy- weeks after operation, the frog showed
pothalamicus and ended deeply within the no behavioral response to the visual stimthalamus (thalamic branch no. 1, fig. 1B). uli of objects brought near or to threat( 3 ) A third grain path separated from ening gestures. Despite this failure to
demonstrate regeneration behaviorally, it
the marginal optic tract just anterior to
was elected to inject the right vitreous
the optic lobe and ended deeply within the chamber.
thalamus i n a cluster of cells just anterior
Histological sections showed that the
to the wall of the ventricle of the optic left optic nerve failed to connect to the
lobe (thalamic branch no. 2, fig. 1C).
brain and did not regenerate i n this single
In general, it was found that the greater specimen. However, the right optic nerve
the interval between injection and sacri- did grow into the right side of the thalafice, the greater became the concentration mus. Figure 3 illustrates a grain path
of grains in the contralateral lobe relative which extended from the regenerated right
to the retina. Whereas the four and one- optic nerve into the brain and immediately
half minute, six hour, one and two day separated into two distinct branches. One
specimens showed far greater concentra- branch (labeled d ) passed ventral to the
tion of grains in the retina than in the preoptic nucleus, near the former chiasm,
contralateral optic lobe, the later speci- crossing over to the left side. It could be
mens showed concentrations of grains in followed for a short distance along the left
the latter area approaching and, i n some marginal optic tract. More anterior secareas, equaling or exceeding retinal con- tions failed to reveal any further extension
centration. The retina was not included of this branch beyond the marginal optic
tract.
with the 32 day specimen.
The second optic nerve branch (labeled
There is some evidence of the existence
of fibers which do not decussate. This is i) remained on the same side, extended
seen in the fact that areas of the contra- directly across to the thalamic ventricle
lateral side characterized by a particularly and then curved dorsally, running along
heavy concentration of grains were often the right lateral border of the ventricle.
matched by corresponding areas of the Sections through the optic lobes revealed
ipsilateral side with a slight increase of slightly greater grain concentrations on
grains However, the much greater con- the right than on the left. Sections through
centration of label on the contralateral the nucleus lateralis tegmenti and second
side indicates that the proportion of non- thalamic branch were unsatisfactory for
interpretation.
decussating fibers must be small.
DISCUSSION
The variability in the treatment of our
specimens precludes the use of the present
In a11 injected specimens, a diffuse, relmaterial for quantitative analysis by grain atively light grain distribution was found
counts. We hope to be able to carry out throughout the brain as well as surroundsuch a n analysis on material in which ing nonneural tissues. This is assumed
treatment has been standardized at con- to represent label spread through blood
ditions indicated to be optimal by the or other fluid movement since the nonpresent study (fixation at over six days injected controls, after a comparable pepostinjection and exposure time greater riod of radioautographic exposure showed
than one month). We found no evidence f a r less diffuse graining in corresponding
of a significant differential concentration areas than any of the injected specimens.
of grains in the hypothalamus on either This fact and the low level of grains in
side nor in the hypothalamus compared to the emulsion that overlies nontissue areas
other regions. Thus, we found n o evidence indicates that the level of background
of direct penetration of the hypothalamus radiation has been kept low enough not to
by optic fibers to provide a possible path- interfere with the detection of either gen-
330
STEPHEN GOLDBERG AND MINORU KOTANI
Fig. 3 Photograph of cross section through brain of young frog with transplanted, regenerated optic nerve. Arrows indicate direction of grain pathways from regenerated optic
nerve; r.o.n., right optic nerve; d, decussating branch; i, ipsilaterd branch, X 90.
era1 or axonal labeling. Within the brain
substance, no difficulty was experienced
in distinguishing pigment from label because the pigment granules were goldenbrown, larger, and, of course, not lying
in the plane of the emulsion.
It was unexpected to find that labeled
material had reached the contralateral optic lobe as early as six hours postinjection.
The distance between retina and chiasm
in the six hour specimen is at least 3 m m
and the distance between chiasm and optic lobe is at least 2.5 m m as measured by
serial sections. Hence, labeled material
traveled to the optic lobe at a rate of at
least 22mm/day if one measures from
the retina, or 10 mm/day measuring from
the chiasm. This rate is out of proportion
to the 1-4 mm/day rates reported by many
authors (Lubinska, '64). Two explanations
can be offered for this:
(1) The label did not travel within the
axon but outside it, along some kind of
tissue plane where it could move faster.
(2) The label did travel within the axon, but axonal flow is much faster in the
metamorphosing tadpole.
Likewise, the grain distribution i n tadpoles sacrificed at later days may represent extraaxonal transfer or intraaxonal
flow. It could not be determined whether
the grains were inside or outside the axons. Electron microscopy may help clarify
this point.
The course of the optic nerve, the marginal optic tract, and the projections to
the optic lobe and nucleus lateralis tegmenti in our experiment appear to correlate well with the pathways described by
Herrick ('17, '25; Kappers et al., '60).
What we have called thalamic branch
number 1 corresponds to the opticoides
Bundel described by Wlassak (1893) in
Rana escubenta. He also described a more
deeply running axial bundle which passes
through both the nucleus anterior superior
corporis geniculate thalami of Bellonci and
the corpus geniculatum of Gaupp. This is
seen in our specimens a s simply a deeper
extension of the marginal optic tract which
merges with the opticoides Bundel to form
thalamic branch no. 1 (fig. 1 B ) . The
authors could find no reference in the
NERVE TRACING VIA RADIOAUTOGRAPHY
literature to the small cluster of cells of
thalamic branch no. 2 (fig. 1C).
The grain pathways followed in the
single specimen with optic nerve transplantation probably represent the course
of the fibers of the regenerated right optic nerve, demonstrating one component
which redecussates and another which remains on the same side. This description
is compatible with the histological findings
in Sperry's experiment ('45).
In order to evaluate accurately the value
of this technique, it will first be necessary
to use it to map out a number of already
well known pathways, comparing results
when using this technique with those of
more classical methods. Further refinements, such as the use of a mixture of
labeled amino acids in a more slowly diffusing form, may yield more detailed information.
ACKNOWLEDGMENTS
The authors wish to thank Dr. W. Etkin,
Dr. R. D. Terry and Dr. J. Padawer for
their advice on and support of this project,
Mr. R. Stern and Mr. S. Weinzimer for
their most valuable recommendations concerning technical details, and Mr. s. Cohen
for the illustrations. The investigation was
supported by National Science Foundation
grant GB-493 to Dr. Etkin, National Institute of Neurological Diseases and Blindness grant NB-02255 to Dr. Terry, and a
student grant from Lederle Laboratories to
S . Goldberg.
331
LITERATURE CITED
1965 The fate of newly synthesized
proteins in neurons. Symp. Internat. SOC.Cell
Biol., 4: 154-175.
Droz, B., and C. P. Leblond 1963 Axonal migration of proteins in the central nervous system and peripheral nerves as shown by radioautography. J. Comp. Neur., 121: 325-346.
Herrick, C. J. 1917 The internal structure of
the midbrain and thalamus of Necturus. J.
Comp. Neur., 28: 215.
1925 The amphibian forebrain 111.
The optic tracts and centers of Amblystama
and the frog. J. Comp. Neur., 39: 433.
Kappers, C. U. A., G. C. Huber and E. C. Crosby
1960 The Comparative Anatomy of the Nervous System of Vertebrates, Including Man.
Hafner Publishing Co., vol. 11, pp. 939-953.
Lubinska, L. 1964 Axoplasmic streaming in
regenerating and normal n e ~ v e fibers. In:
Mechanisms of Neural Regeneration. Progress
in Brain Research, M. Singer and J. P. Schadk,
eds., vol. 13, pp. 1-72.
Singer, M., and M. M. Saltpeter 1966 The
transport of tritium labeled 1-histidine through
the Schwann and myelin sheaths into the axon
of peripheral nerves. Anat. Rec., 154: 423.
Sperry, R. W. 1945 Restoration of vision after
crossing of optic nerves and after contralateral
transplantation of eye. J. Neurophysiol., 8:
15-28.
Turbes, C. 1965 Centripetal movement of am&
no acid protein complex in the visual system.
Anat. Rec., 151: 427.
Weiss, P. 1965 Synthesis and flow of neuroplasm: a progress report. Science, 148: 669670.
Wlassak, R.
1893
Die optischen Leitungsbahnen des Frosches. Archiv. f. Anat. u.
Physiol., Physiol. Abt., Supp1.-Bd. S. 1-28.
Droz, B.
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