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AMPHIBIAN EAR VESICLE AND FURTHER
EXPERIMENTS ON EQUILIBRATION


By


George L. Streeter, M.D.
B y


George L. Streeter, M D.




 
REPRINTED FROM
reprinted from


THE JOURNAL OF EXPERIMENTAL ZOOLOGY  
THE JOURNAL OF EXPERIMENTAL ZOOLOGY  


Volume III
Volume IV
No. 4
No. 3
 


BALTIMORE, MD., U. S. A
December, 1906


BALTIMORE, MD., U.S. A.
September, 1907






From the Anatomical Laboratory of the Johns Hopkins University




SOME EXPERIMENTS ON THE DEVELOPING EAR
VESICLE OF THE TADPOLE WITH RELATION ,


TO EQUILIBRATION*


BY
ii M\ ^ ^ IXC '


GEORGE L. STREETER, M.D.


Associate, Wistar Institute of Anatomy  
From the Wistar Institute of Anatomy and Biology, Philadelphia
With Twelve Figures


The eventual object of the experiments reported in the following
paper was the rearing of some tadpoles which had been deprived
of their auditory vesicle and acoustic ganglion, either on one side
alone or on both sides; that is to say, an artificial production of a
unilateral and bilateral absence of the acoustic apparatus. This
was done in the expectation that it might be possible to trace the
central acoustic path, in this new way, and perhaps throw further
light upon its course and relations. The absence of these sense
organs, however, produced such definite abnormalities in the
behavior of the growing larvae and in the development of their swimming abilities that it became at once apparent that I was dealing
with valuable evidence in respect to their function and its bearing
on the mechanism of equilibrium. It is, therefore, deemed advisable
to restrict the following paper to the physiological features of these
experiments, and reserve the study of the central nervous system
of the reared specimens for a later communication.


What we already know concerning the function of the vertebrate
SOME FACTORS IN THE DEVELOPMENT OF THE
ear is based principally on experimental sectioning or stimulation of the semicircular canals, or the nerves to their ampullae, in
AMPHIBIAN EAR VESICLE AND FURTHER EXPERIMENTS ON EQUILIBRATION
adult birds and fishes.^


^Read in part before the Section of Anatomy of the British Medical Association, at the meeting held
i KY
in Toronto, August 21-25, ^906.


^For experimental work on fishes we are for the most part indebted to Lee (’93 and ’98) and Lyon
GEORGE L. STREETER, M.D.  
(’00), both of whom carried on their experiments at the Woods Hole Laboratories. Further work on
fishes has just been completed at the same place by Professor Parker, whose paper I am told is now in
press and will appear in the Bulletin of the U. S. Fisheries Bureau. An abstract of part of his
work was read before the American Zoological Society (Parker, ’05). A voluminous literature exists
concerning experiments on higher vertebrates, particularly the pigeon, but it need not be considered
here.
 
The Journal of Experimental Zoology, vol. 111, no. 4.
 
u t ^ SI
 
 
544
 
 
George L. Streeter


Associate Professor of Neurology at the Wistar Institute
With Six Figures


The fact that it is possible to experiment on the embryo and to  
In a previous paper concerning experiments on the developing
produce at will practically a congenital absence of thisorgan, besides
ear vesicle 1 it was shown that the group of cells forming the primitive epithelial ear cup or ear vesicle of the tadpole is specialized
serving as a control over the experiments on adult animals, introduces a direct advantage both as regards the ease with which the  
to that degree that although removed to an abnormal environment the cells still continue to differentiate themselves into a structure possessing many of the features of a normal labyrinth. Recently it has been shown by Lewis 2 that even earlier, while still an
operation is performed and as regards its completeness and permanence and freedom from injury of adjoining structures, the latter
uninvaginated plate, the ear anlage is already capable of a certain degree of independent differentiation. In the following paper
point being of particular importance to those who are still in doubt
additional evidence will be given of the high degree of developmental independence possessed by the early labyrinth cells. It
. as to how much is due in the experiments on adults to injuries and
will be pointed out that individual parts of the vesicle may develop
stimulationsassociated with the operation and how much is purely
independently of the rest of the vesicle. It will also be shown that
the result of the cessation of the stimuli which normally originate in  
the process of differentiation extends to the difference existing
the labyrinth. Furthermore, since the labyrinth is removed during
between a right and left-sided organ. A left ear vesicle transplanted into the empty pocket left by the removal of the right ear
the early formative period at a time when it may be presumed that
vesicle develops into a labyrinth that is perfect in general form
the various organs possess their greatest adaptability, it will be seen
and in its relations to the brain, with the exception that it maintains its left-sided character; the anterior semicircular canal is  
that such embryonic interference affords a most complete test
found on the caudal side toward the vagus group, while the posterior canal lies toward the eye, and likewise the lagena which
of the power of functional compensation on the part of other
organs.


Behavior of Normal Tadpoles
1 Streeter, G. L., *o6: Some experiments on the developing ear vesicle of the tadpole with relation
to equilibration. Jour, of Experimental Zool., vol. iii.


In analyzing the behavior of operated specimens it was found
2 Lewis, W. H., ’07: On the origin and differentiation of the otic vesicle in amphibian embryos.
necessary to make a preliminary study of control tadpoles, in order
Anatomical Record, No. 6, Amer. Jour, of Anat., vol. vii.
to determine the normal development of motor reflexes and their
coordination and the consequent establishment of equilibrium.
This was done by removing the larvae from their gelatinous capsule shortly after fertilization and following their development in  
tap water. In this way it was seen that in the process of learning
to swim they pass through three periods, which may be named as
follows:


1. Stage of non-motility, first three days.  
The Journal of Experimental Zoology, vol. iv, no. 3.  


2. Stage of spinal reflexes, fourth to sixth days.
(5 1 & 3


3. Stage of equilibrium, sixth day to maturity.


The first stage, with a favorable temperature, lasts from the
43 2
time of fertilization to the third or fourth day. During this time
the larvae, aside from the movement due to cilia, lie motionless on
their side on the bottom of the dish and do not respond to stimuli.
The second stage begins at the time when they first respond to




Experiments on the Developing Ear Vesicle
George L. Streeter , M.D.




545
normally buds out from the caudal border of the saccule in these
cases is found extending forward toward the prootic ganglion.


The ear vesicle, however, is not in all respects independent of
the surrounding structures. Some experiments which are reported
below, indicate that its position in reference to the brain, ganglion
masses and the surface of the body is determined by the environment itself; it may be rotated in any direction, and nevertheless it
eventually develops in the normal attitude, with the saccule toward
the ventral surface, the semicircular canals toward the dorsal
surface, the lateral semicircular canal being toward the lateral
surface, and the endolymphatic appendage toward the brain.


mechanical stimuli by flexion of the body and taild These reactions consist of simple motor reflexes at first, but they soon become
The experiments were carried out on larvae of Rana sylvatica
combined and coordinated so that by a series of such body flexions
and Rana pipiens, and the operating stage was the same that
they are able to wiggle rapidly forward on the bottom of the dish.  
was used in previous experiments. 3 The time is just at the close
This manner of progression evidently consists entirely of spinal
of the non-motile stage, and the epithelial ear consists of an invaginated cup-shaped mass of cells just in the process of being pinched
cord reflexes and is not controlled by higher centers. In order to
off from the deeper layer of the skin, with the edges turning in to
perform it, it is necessary for the tadpoles to touch the bottom or
form a closed vesicle. For simplicity the term “ear vesicle” will
be used even though the closure is not yet complete; the attempt
to distinguish between auditory cup and auditory vesicle does not
seem to be justified for the present purposes. The technique of
the operations was also the same as that described in the previous
paper. Notes were made on the behavior of the animals, and
at the end of from four to six weeks the specimens were preserved
in a chrome-acetic mixture, cut in serial sections, and stained with
haematoxylin and congo red. With certain specimens the ear
vesicle, adjacent ganglia, and a portion of the central nervous system were reconstructed after the Born wax plate method. Eleven
such models were made, and photographs of some of them are
reproduced in Figs. 2, 3 and 6. With the aid of these models it
was possible to identify relations and detailed features of the labyrinths that otherwise could not have been recognized.


The morphological features of the experiments will be first considered, and the behavior of the animals and its relation to equilibrium will be treated separately in the latter part of the paper.




Streeter ’06: /. c., Fig. 3, p. 547.


Fig. I, Outline drawing of normal tadpole (Rana sylvatica) of the second stage or stage of spinal
reflexes. Enlarged 8 diameters.


Fig. 2, Outline drawing of normal tadpole (Rana sylvatica) at the beginning of the third stage. This
Development of Amphibian Ear Vesicle
specimen had the power of equilibration, although sections of the ear vesicle showed that the
development of the semicircular canals was not yet complete. Enlarged 8 diameters.


side of the dish; when they are driven up into the free water with
a pipette, where there is no contact with solid objects, they make
no effort at movement, but sink inertly to the bottom; on striking
the bottom they run forward again. The third stage begins when
they are first able to move freely about without touching solid


^ I have been informed by Dr. R. G. Harrison that it is just at this time that the motor nerve
433
roots make their appearance, and this may determine the onset of the second stage. According to
his observations the power of muscle contraction follows almost immediately after the development
of the motor roots; but it never precedes their development, as is maintained by some. He has
found the motor root present in specimens that had not yet moved.




DETERMINATION OF POSITION OF THE EAR VESICLE


The conclusion that the attitude of the developed labyrinth, the
position of its canals and various chambers, is determined by its
environment is based on seventeen experiments in which the ear
vesicle was loosened from its normal situation and placed in an
abnormal attitude, and the specimen then allowed to continue in
its development. At the end of a month examination showed that
the labyrinth had become differentiated with varying degrees of
completeness, and in each instance had developed in normal relation to the surrounding structures.


Rotation in Two Directions. In eight of these experiments the
ear vesicle was rotated i8o° around both its vertical and transverse
axes, so that it was turned face inward and upside down ; or, in other
words, its lateral or invaginated surface was toward the brain and
its ventral border was where the dorsal border should be, the maximum displacement. After this procedure the wounds healed
within a few hours, and the larvae were reared up to the fourth or
fifth week, when they were killed and cut in serial sections. The
labyrinths of five specimens were reconstructed. Before describing them reference should be made to the normal condition of the
labyrinth at this age. A reconstruction of a normal one with its
adjacent structures is shown in Fig. I.


From the reconstruction of a normal specimen it can be seen that
the three semicircular canals have individual characteristics by
which they can be separately identified; such as the Y-shaped
union of the anterior and lateral canals, and the overlapping of
the caudal end of the lateral canal by the posterior canal, and the
junction of the posterior and anterior canals to form the crus commune. The differentiation between utricle and saccule is not yet
complete, but the part that is to become saccule is so labeled.
From the caudal border of the saccule can be seen a small pocket
budding out which constitutes the lagena or primitive cochlea.
Directly median to the crus commune is the endolymphatic appendage, consisting of a small duct leading from the main labyrinth
chamber up between the labyrinth and brain to a rounded pouch,
the saccus endolymphaticus. In their histology, as well as in




434




George L. Streeter , M.D.




their general form, the various parts of the labyrinth exhibit at
this time individuality. (See Fig. 4.) The ventro-median portion
of the vestibular sac and the ampullar ends of the semicircular
canals possess high columnar cells forming the neuro-epithelial
maculae which are supplied with fibers from the acoustic ganglion,
lying against the medial wall of the labyrinth. The endolymphatic
sac has cuboidal cells, and the lagena has intensely staining columnar cells like those seen in the macular regions. The lagena
is further characterized by its sharply rounded outline, and by the
fact of its being compactly surrounded by ganglion cells and fibers,
and cartilage forming cells. These features are so definite that


Crus commune






Fig. 1 Reconstruction showing the form and relations of the membranous labyrinth of a normal
tadpole (Rana pipiens) one month old. The labyrinth, adjacent ganglia and part of the brain w.. re
reconstructed after the Born method, and the remainder of the figure was drawn from a dissection of a tadpole of the same age. Enlarged 35 diameters.


54 ^ George L. Streeter
the various parts of the labyrinth can be recognized without difficulty, even though they happen to be incomplete, or out of their
normal relations.  


objects. At this time a new control over their movements is developed, in virtue of which they become able to leave the bottom of the  
Now if one examines the models of the operated specimens,  
dish and swim up into free water with maintenance of what may
photographs of three of which are reproduced in Fig. 2, it is seen
then be called equilibrium. The form of the tadpole during the  
that the individuality of the semicircular canals can at once be
latter part of the first stage is shown in Fig. 3. The second and
identified. In model a , the canals are practically normal; in model
third stages are shown in Figs, i and 2.
b, the anterior canal is small, and the lateral canal consists only
of a pouch which has not been pinched off from the main cavi-ty;
in model c , the posterior canal remains a simple pouch, while the


The correlation between the histological development of the
labyrinth and the development of the power of equilibrium was
studied by selecting specimens of the second and the beginning of
the third stages, carefully noting their behavior, and then cutting
them in serial sections.^


From these series it could be seen that shortly before the animal
Development of Amphibian Ear Vesicle
enters the stage of equilibrium the labyrinth consists of a closed
epithelial sac incompletely subdivided into compartments and
possessing differentiated nerve endings which are connected with
the brain by the acoustic nerve and ganglion. That at least one
such apparatus is essential for equilibrium will be seen when I
describe the behavior of tadpoles that have been completely
deprived of the same. As regards the semicircular canals it is a
different matter; they can already be seen in the process of
development, but are not completely pocketed off until after equilibrium is already established. Consequentlythe semicircular canals
as such are not an essential factor in equilibration.


Method of Operation


Larvap of Rana sylvatica measuring about 3 mm. long were
435
selected as being most suitable for the operation. Their general
form at this time is shown in Fig. 3. There is a distinct tail bud,
and on the head the eminences caused by the optic cup and head
ganglia are visible. The structure that is to form the future labyrinth is situated just dorsal to the ganglionic eminence and is shown


^ The correlation between the histogenesis of organs and the development of their functional activity
forms a fruitful field which has been explored by comparatively few investigators. It may be approached
both through ontogeny and phylogeny. Prentiss (’oi) by this means worked out important facts
regarding the crustacean otocyst. Many details concerning the verte brate ear which do not belong to
the scope of the present paper could doubtless be learned in the same way.


anterior and lateral canals are normal. In considering the posture
of the canals it is to be noted that the surrounding structures have
been left out in Fig. 2, to avoid unnecessary duplication; the three
models are all represented in the same relative position as that of
the labyrinth in Fig. 1, 1. e., the cephalic end is on the right, the
caudal end is on the left, the ventral surface is below, and the dorsal surface is above. Thus it will be seen that the lateral canals
in all three models are in the same plane; likewise the posterior
canals all form the dorso-caudal border of the labyrinth, and the
anterior canals form the dorso-cephalic border. The fact that
the anterior canal is small in model b , 4 and the posterior canal is
small in model c , gives rise to a false impression of a backward


Experiments on the Developing Ear Vesicle


sac. endolymph.


547




in Fig. 3 by the mark +. It consists of a cup-shaped mass of
laeena
cells (auditory cup) which have differentiated themselves from
the deeper layer of epidermis, and are just
in the process of closing in at the edges to
form the completed ear vesicle. In size
this ear cup or ear vesicle is about one-
half that of the optic cup.


For performing the operation it is not
a h b C
necessary to anesthetize the specimen as
it is still in the non-motile stage and does
not respond to stimulation. After removing the larva from its gelatinous capsule it


and an incision made near the place indicated in Fig. 3. The edge of the incision
is then raised a little and the auditory cup
is picked out with a needle. After a little
practice one learns to make the incision directly at the edge of the
cup so that it comes away easily and intact, resembling somewhat
a thimbleberry. Lying just in front of it is the acoustic ganglion
which is not so sharply outlined. This is also removed and,
in order to make sure that it is all taken out, the surrounding
mesoderm is cleaned out as far in as the brain. Where but one
vesicle is to be removed the operation is then complete, and the
specimen is left to proceed in its development. The wound
immediately closes of itself and heals in the course of a few hours
leaving no trace of the operation. Where both sides are operated
on, the same procedure is carried out on both sides. The ear
vesicle never regenerates following complete removal.


The ear vesicle was removed on one side from thirty specimens
Fig. 2 Reconstructions showing the form and posture developed by three labyrinths one month old’
and on both sides from twenty specimens. The animals were then
which while primitive ear vesicles were rotated from their normal position so as to lie face inward and
kept under observation and their behavior recorded through the  
upside down. The models are placed so that their planes are parallel with those in Fig. I. Thus they
whole larval period and until the completion of metamorphosis.  
present a lateral view with the cephalic end toward the right, caudal end toward the left, dorsal surface
The following notes were selected from these records.  
above, and ventral surface below. Enlarged 50 diameters.  


and forward tilting of the vesicle. The saccule and lagena have
the same position as in Fig. i, and the lagena points caudally as
it should do. The endolymphatic appendage lies on the median
side of the crus commune; in models b and c it is small, but the tip


is placed under a binocular microscope
4 This may be due to injury received at the time of operation. Such localized defects are frequently
seen. They may involve any part of the labyrinth, and they vary greatly in the extent of the labyrinth
wall affected. In one case the entire labyrinth was defective, with the exception of the endolymphatic
appendage, which was normal in structure and position, and presented a curious appearance, being
attached to the small irregular vesicle representing the labyrinth. Such localization of abnormal
development is evidence of the high degree of specialization of the cells forming the primitive ear vesicle.






Fig. 3. Outline drawing of Rana
43 6
sylvatica at the time suitable for
operation, just at the end of the non-
motile stage. The tail bud is present
and on the head are seen the eminences due to the optic cup and head
ganglia. Above the latter is the
point of operation shown by a cross.
Enlarged 8 diameters.




George L. Streeter , M.D.




54 ^ George L. Streeter
of it can be seen in model a. The acoustic nerve and ganglion are
attached to the median and ventral surfaces of the labyrinth, and
the nerve connection with the brain appears to be normal.  


Removal of One Ear Vesicle
The conditions found in the three specimens pictured in Fig. 2
are typical of what is found in the other five specimens examined.
They vary in the completeness of their differentiation, some of
them consisting of only a vesicle with perhaps a single canal pouch,
but in all cases the acoustic ganglion is present on the ventro
medial surface, and the macular areas can be recognized. The
lagena is present in seven out of eight cases. The endolymphatic
appendage developed in six out of eight cases. As regards posture,
the rule is that the more perfectly the labyrinth is developed the
more accurately its posture corresponds to the normal relations.
But even in the most imperfect specimens when the endolymphatic appendage appears it is on the medial surface, and the tendency to canal formation is always on the dorso-lateral surface,
and the saccule and lagena appear on the ventral surface. This
condition of course applies only to vesicles that have been implanted in the acoustic region as was done in all the above cases.


Twenty-four hours after operation: Specimens are 5.5 mm. long
Rotation in One Direction. In four experiments the ear vesicle was rotated 180° around its vertical axis, i.e> turned face inward.  
and show presence of gill buds. In appearance and behavior^ no
These specimens were then reared as in the preceding instance,
difference can be detected between them and normal tadpoles.  
and eventually cut in serial sections. A reconstruction model of
They lie on their side and on stimulation flex their body, but make
one of them is reproduced in Fig. 3, and if it is compared with
no attempt at swimming.  
Fig. 1 it will be seen that although the vesicle was started in its
development with invaginated side toward the brain yet the completed labyrinth has the normal posture. A section of the same
specimen is reproduced in Fig. 4, showing the labyrinth surrounded
by developing cartilage. The acoustic ganglion is connected
in normal manner with the brain and sends peripheral fibers to
the thickened floor of the saccule. The endolymphatic sac is in
its normal position, and the narrow duct can be seen connecting
it with the main chamber of the labyrinth directly median to the
crus commune. The series through this specimen show that histologically it is practically perfect. Of the other three specimens
one was almost equally perfect, another showed some abnormalities in the formation of the canals and the lagena, and the


Forty-eight hours after operation: Specimens are 7 mm. long,
gills are branched and the blood can be seen circulating through
them. In appearance and behavior they still show no departure
from that seen in normal control specimens. While at rest they
lie on theii side. On stimulation (sunlight, jarring the dish, or
touching with needle) by a rapid flexion of the body and tail from
side to side they swim forward, 5-10 cm., on the bottom of the
dish in a straight or slightly curved line, and then come to rest on
their side, and remain so until a new stimulation excites another
such excursion. Their course is directed either by the side or
bottom of the dish. When forced up into free water the flexions
stop and they sink inertly to the bottom.


Third day after operation: Specimens average about 8 mm.
Development of Amphibian Ear Vesicle
long, abdominal epidermis differentiated from that of the dorsal
parts of the body by being less pigmented. Appearance and behavior is still practically normal. They begin to show a tendency
to assume the upright position while at rest, but no great importance can be attached to this feature as throughout the early days
of the tadpole period, preserved specimens lie in the same positions
as living ones. Their posture in water may be entirely determined
by their body proportions. Their movements remain of the spinal
cord type seen on the previous day, the response being more prompt.


Fourth day after operation: Specimens 9-9.5 mm. long. In
appearance the operated specimens are the same as the normal
ones, but in behavior they present a difference. The normal ones
still confine their movements to the bottom»or side of the dish;
when stirred up into free water, though most of them still roll about
inertly, some of them are able to maintain a direct course. On the
other hand the operated ones, as soon as they are driven from the
bottom, swim in a spiral or circular manner as shown in the


437


Experiments on the Developing Ear Vesicle


fourth was quite imperfect, consisting of only a large vesicle with
a thickened epithelial floor connected by a few nerve cells and
fibers with the brain.


549




accompanying Fig. 4. The tendency is to swim with the operated
Fig. 3 Reconstruction of a tadpole labyrinth one month old, which when a primitive ear vesicle was
side under, and in the rolling movements around the long axis of  
rotated from the normal position i8o° in one direction, so as to lie with invaginated side toward the brain.
the body it is from the operated side under to the opposite. When
A section through the same labyrinth is shown in Fig. 4. Enlarged 55 diameters.  
these same specimens touch the bottom they are able to direct their
course as on the previous two days. Evidently, a functional union
is normally established at about this time between the ear vesicle  
and the spinal cord reflex centers, upon which the individual is
dependent for maintafning its position in free water, and it is not
until this occurs that the removal of the ear vesicle causes any
symptoms.  






Fig. 4 Section through the membranous labyrinth shown in Fig. 3. It shows that though originally
turned face inward it has developed in the normal attitude, e, endolymphatic appendage; c.c., crus commune; sacc., saccule; c. lat., lateral semicircular canal; gang, acust., acoustic ganglion. Enlarged
55 diameters.


Fig. 4. Sketch showing three typical swimming movements made by specimens on the fourth day
Transplanted Specimens. The irregularity of form of the six
after removal of their left ear vesicle.
specimens transplanted to the region between the eye and nostril,
previously reported , 5 is so great that they give no assistance in solv


Sixth day after operation: Specimens about 12 mm. long, and
Streeter ’06, l. c., p. 557.  
have commenced to nibble at food and pass faeces. The characteristic movements which first appeared on the fourth day have
become stronger and stand out in more marked contrast to the
behavior of normal specimens which at this time can swim easily
up into free water with accurate maintenance of equilibrium.  


Seventh day after operation: The operated specimens show
distinct improvement in swimming ability; many of them are now
able to maintain a fairly direct course in free water, but on excitation they renew the spirals and circles which characterized the
fourth, fifth and sixth days.






43 8




George L. Streeter , M.D.




550
ing the question of posture. However, in five cases, which will
be presently described, where the ear vesicle was transplanted
from the left side to the right side into the place made vacant by
the removal of the right ear vesicle, in spite of the fact that these
ear vesicles were implanted with haphazard attitude toward the
adjacent structures, they nevertheless in each instance, developed
right-side up, and with the median surface toward the brain, as
can be seen in Figs. 5 and 6.




George L. Streeter
Mesen


Sac.ehdolymph j


Eighth day after operation: nearly all the specimens now
swim freely and directly in all parts of the water, and irregularity
of swimming is only elicited by excitement.


Tenth day after operation: Swimming is practically normal.  
ce p h.  
Their movements are under such control as to enable them to support themselves in free water and nibble at floating stems and
Dience p h.  
leaves. It can be seen, however, that in swimming they lean






Fig. 5. Photograph of a frog whose left ear vesicle was removed when a tadpole 3 mm. long. The
Fig. 5 Reconstruction showing the form and relations developed by a left ear vesicle when transplanted to the right side; it shows that under such circumstances the ear vesicle retains its left-sided
only asymmetry noticeable is the absence of the ear elevation on the left side normally caused by the
characteristics, though it otherwise normally adapts itself to its new situation. A photograph of the  
labyrinth and its cartilaginous capsule; the lateral line on that side is straight from the eye back, while
same specimen is shown in Fig. 6, c.  
on the right or normal side it is deflected. The posture is normal. Enlarged 3^ diameters.  




slightly toward the operated side, a sympton which persists throughout their larval period.
DETERMINATION OF THE D EXTRAL AND SINISTRAL CHARACTER
OF THE EAR VESICLE


Twelfth day after operation: Specimens are normal as regards
The question as to whether the right or left-sidedness of the ear
size, nourishment, and symmetry, except for the absence on the  
labyrinth is controlled by the environment, or is determined by
operated side of the elevation which is caused normally by the
some intrinsic character of its own constituent cells, is answered
labyrinth and its cartilaginous capsule. In behavior they differ
in favor of the latter by the fact that if the left primitive ear vesicle,  
from the normal only in the slight leaning toward the operated side
before the time of its complete closure, is transplanted to the opposite side of the embryo it retains its original left-sidedness. In five
specimens, at the usual operating stage, the right ear vesicle was
removed, and at the same time the left ear vesicle was uncovered
and lifted from its natural bed and then placed into the pocket




Development of Amphibian Ear Vesicle




439


Experiments on the Developing Ear Vesicle 55 ^


and a momentary loss of equilibrium which can be elicited by
from which the right vesicle had been taken and allowed to heal.
excitement.  
In making the transplantation no effort was made to place the
ear vesicles in any particular posture. After keeping the specimens alive for one month they were sectioned and from three of
them reconstructions were made of the transplanted ear vesicle
together with the adjacent structures. The three labyrinths are
shown in Fig. 6, and model c is again shown in Fig. 5, with the
brain included. It will be seen that in developing they have
assumed the normal attitude toward the brain. The endolym


Three months after operation: The specimens passed through
sac. endolymph.  
a normal metamorphosis at the end of the third month. A photograph made of one of them a few days after the completion of the
process is shown in the accompanying Fig. 5.  


As long as they continued as swimming tadpoles the slight leaning toward the operated side persisted and it was possible through
excitement to cause a momentary disturbance in equilibrium, but
the latter became gradually more difficult to demonstrate. As
soon as they commenced to make use of their legs the character
of the swimming changed; it then became a series of leg strokes
instead of the sinuous flexions of the body and tail. After that it
was no longer possible to detect the leaning toward the operated
side; both when swimming and when at rest their behavior was to
all appearance normal. When taken out of water they jumped
normally and came to rest in a normal posture. When turned
over on their backs they righted themselves promptly.


The fact that the slight disturbance of equilibrium, which could
be still detected in the tail-swimming tadpole, could no longer be
seen in the leg-swimming frog, a change completed within four or
five days, probably does not signify the cure of the condition, but
rather that under the latter circumstances a slight defect is more
difficult to recognize. The corollary of this would be that equilibrium in the swimming tadpole is a more delicately balanced
mechanism than in the kicking and jumping frog.


lagena


Removal of Both Ear Vesicles
a be


During the first three days after the operation the appearance
and behavior of these specimens are the same as seen in the normal
ones, and in those from which one ear vesicle was removed. The
response to stimuli is perhaps a trifle less prompt, but otherwise
they could not be distinguished one from the other.


Fourth day after operation: It was seen that in one-sided operations the specimens commenced about this time to make excur-
Fig. 6 Reconstructions of three labyrinths which while primitive ear vesicles were transplanted
from the left to the right side. They are all represented in the same position as the models in Fig. 2.
The model c is the same that is shown in Fig. 5. Enlarged 50 diameters.


phatic appendage and the median side of the labyrinth is toward
the brain, the semicircular canals are toward the dorso-lateral surface, and the saccule and lagena are toward the ventral surface.
But it can at once be recognized that the saccule and lagena point
forward toward the eye, and that the anterior and posterior canals
are in reversed positions. We thus have a complete mirror image
of the right labyrinth, i. e a left labyrinth. Model a possesses
three semicircular canals and is almost a normally formed left
labyrinth. In model b the lateral canal consists of a pouch whose
walls did not undergo the customary approximation and central
absorption. In model c the posterior canal is not pinched off.
In each of the models the lagena, saccule, and endolymphatic


552




George L. Streeter
440




sions into free water, and in doing it they departed from the normal by swimming in spirals and circles. Tadpoles with both ear
George L. Streeter , M.D.  
vesicles taken out make no such excursions and show decidedly*
less activity. Occasionally they flex their body and tail from side
to side producing a snapping effect which does not result in any
forward progress. Like the other specimens they are, however,
able to wiggle along in contact with the side and bottom of the
dish. *


Seventh day after operation: The specimens are smaller and
are retarded in development as compared with the normal and
one-sided specimens. They are, however, symmetrical in form
and are normal as regards the appearance and movements of the
eyes, mouth, heart and intestine. They are decidedly less active
and stimuli produce irregular attempts at swimming, sometimes
somewhat spiral in character but usually nothing more than a
series of awkward flexions of the body. These flexions also
occasionally occur with no apparent stimulus. They make a
partially successful effort at nibbling on the bottom of the dish.


Twelfth day after operation: Absolutely no improvement in
appendage are typical, and there is establishment of normal
swimming; any attempt at it results in a series of somersaults,  
appearing nerve and ganglion connections.  
they throw their body up into the water and then promptly sink
to the bottom in almost any position. When at rest, they lie on
their side, back, or normally on their belly, depending apparently
on whether their intestine is filled with sand, etc., to properly
balance the body. The intestine is very apt to be empty because
of the difficulty they experience in feeding. They do not wiggle
along on the bottom as well as they did on the fourth and fifth
days.  


Two months after operation: The specimens could not be
EQUILIBRATION
carried much beyond this point, the difficulty apparently being
starvation from inability to wander around and collect food.
Perhaps also the respiration was involved, for they were unable
to go to the surface for oxygen as the normal tadpole does.


In behavior they show no improvement. For the most part they
It was found in the experiments performed a year ago that
lie stiff and inert in various positions on the bottom, and their
removal of one or both ear vesicles, just after they are pinched off
occasional attempts at swimming have never developed into anything
from the skin, produces in the tadpoles definite disturbances in
more successful than was described on the seventh and twelfth
the development of their power of equilibration. It was found
that when a tadpole is deprived of but one ear vesicle he is by
virtue of the remaining one able to develop practically normal
swimming abilities; but when both ear vesicles are removed the  
results are more serious, and in that case the tadpole never develops any sense of equilibrium and is never able to swim. The
loss is not compensated for by any other organ and the animal
lies helpless on the bottom of the dish. With one ear vesicle the
tadpole swims practically in normal fashion, and with no ear vesicle he cannot swim at all.


The fact that one ear vesicle is sufficient for the maintenance of
equilibrium greatly simplifies the study of this mechanism; it
means that one side can be immediately eliminated, and the problem is reduced from a bilateral one to a unilateral one. A series of
experiments at once suggested themselves, in which the ear vesicle of one side was to be removed, and then various operative
procedures undertaken upon the ear vesicle of the opposite side,
and the test of its consequent functional ability was to be the very
decisive one of whether the animal could swim properly, or
whether it could not swim at all.


Experiments on the Development Ear Vesicle
In the paper referred to there is described the experiment of
transplanting the ear vesicle into a subdermal pocket in from of
the eye. When this was done the transplanted ear vesicle continued
in its development, and in some instances established a nerve-ganglion connection with the forebrain; but such specimens never gave
evidence of functional activity. The failure to functionate was
not unexpected, inasmuch as the connections established were
at an abnormal situation, and furthermore the vesicles though
having developed many essential features of the normal labyrinth




553
Development of Amphibian Ear Vesicle




days after operation. Their appearance departs from the normal principally in the small contracted character of the abdominal
441
region. In volume they are about one-third as large as the noimal
specimen, varying from 2.5 to 4 cm. in length. They have a hind
leg bud 2.5 to 3 mm. long. As some of them commenced to
die at this time the rest were put in preserving fluids for micro-
scopial purposes.


A summary of the above notes on the operated iindividuals may
perhaps be best formulated by making the following comparison
with the three stages of normal behavior.


First stage: The operation was performed during the latter
were still quite imperfect in the formation of the separate chambers and the semicircular canals. So this year in carrying out the
part, while the animals were still non-motile.  
experiments described in the first part of the present paper the
behavior of the specimens was eagerly watched, and the endeavor
was made to determine the amount of alteration in position and
defectiveness in form that is compatible with functional activity,  
involving the problem of the correlation between function and
morphology. The observations made in the different experiments
have been arranged and condensed as follows:


Second stage: During this period they behave exactly like
a Left ear vesicle removed; right ear vesicle loosened from
normal specimens, both those having one vesicle removed and  
skin and rotated, in six specimens around the vertical axis 180°
those that have been deprived of both vesicles. They respond to
and in eight specimens around both the vertical and transverse
stimuli and learn to wiggle along in contact with the bottom of the  
axis 180 0 . As has already been shown these ear vesicles developed
dish in the normal manner.  
into labyrinths of varying degrees of perfection, some being completely normal in form and having apparently normal ganglion
and nerve connection with the brain wall. (See Figs. 2, 3 and  
4.) The behavior of all the specimens was uniform, both where
the ear vesicle was rotated in one plane and where rotated in two
planes; at the end of a week after the operation, when with a normally functionating labyrinth they should be able to swim freely
and directly, they instead exhibit only irregular movements or spin
around in spirals or circles. Their incoordinate movements continue, and at the end of a month there is no improvement; i.e 9 they
behave exactly like specimens with both ear vesicles removed.
Evidently ear vesicles thus treated do not perform their natural
function.  


Third stage: It is at the beginning of this period that they
b Left ear vesicle removed; right ear vesicle fragmented by
depart from the normal. It can be plainly seen from their conduct
teasing between the points of two needles, the fragments left in  
that something has happened to that controlling influence from'
place. Ten specimens were treated in this way, and were kept  
above, which they require in order to leave the bottom and to swim
under observation four weeks, during which time they gave no
and maintain their position in free water. In case but one ear  
evidence of any sense of equilibrium.  
vesicle is gone they swim in spirals, circles, or straight while rolling around their long axis. This, however, lasts only a few days
and then it is gradually overcome. From then on they swim
almost perfectly; there maybe a slight tilting toward the operated
side and on excitement a momentary loss of equilibrium, but this
would only be seen on careful examination. It is a different matter where both labyrinths are absent; the animals in that case are
completely and permanently incapacitated for swimming. There
is no apparent sense of equilibrium and they never develop any.
The animals were kept alive about two months, at the end of
which time their movements were as irregular as at the beginning.  


Transplantation of Ear Vesicle After Bilateral Removal
c Right ear vesicle removed; left ear vesicle transplanted to
the empty pocket on the right side. Five specimens were operated upon and observed for one month, at the end of which time
they were cut in serial sections, and it was found that the ear vesicles had developed into fairly complete labyrinths, but had maintained the characteristics of a left-sided organ. (Figs. 5 and 6.)


From the above experiments it became evident that a tadpole
having but one labyrinth proceeds in its general growth and


442


554


George L. Streeter , M.D.


George L. Streeter


Throughout the whole period of observation they had exhibited
incoordinate movements, and at the end of that time they were
unable to swim. This and the two previous operations indicated
that rotation of an ear vesicle, or transplanting it from one side
to the other, or fragmenting it was not compatible with the development of its function, in spite of the fact that the ear vesicle proceeded in its development and had become to all appearances
almost a perfect labyrinth. In the next experiments less severe
treatment was tried.


develops swimmingabilities about as well as the normal animal; but
d Left ear vesicle removed; right ear vesicle uncovered and  
specimens deprived of both ear vesicles never learn to swim and  
carefully lifted out and then immediately placed back in its original position, the effort being made to do a minimum amount of  
never develop any sense of equilibrium. The next step was to
injury. Of six specimens all exhibited symptoms of the absence
see if it would be possible to remove both vesicles and atthe same
of all sense of equilibrium.  
time transplant one of them into a new position, having in mind
the successful results obtained by Lewis (’04) in transplantation of  
the optic cup.  


After that operation if the tadpole succeeded in developing
In the experiments a, b , c and d there was the possibility of  
equilibrium and the power of swimming then it would prove that
injury to both the nerve-ganglion connection and the ear vesicle.
a transplanted ear vesicle could establish new connections with the  
In the following experiments the effort was made to restrict the  
central nervous system and develop its normal functions; the ship
injury to one or the other. 1
would simply be sailing with its compass set up in a different place.  


e Left ear vesicle removed; right ear vesicle uncovered and
a fragment cut from the cephalic portion of its wall, care being used
not to otherwise disturb the vesicle. Eight such specimens were
kept five weeks, and none of them developed any sense of equilibrium, or were able to swim.


f Left ear vesicle removed; right ear vesicle uncovered and
a small piece cut from its caudal border, any further disturbance
being avoided as in e. Eight specimens were operated upon, and
after keeping them four weeks none of them could swim properly.


Fig. 6. Tadpole showing elevation in front of eye caused by the transplanted left ear vesicle, the  
g Left ear vesicle removed; longitudinal incision made through
right ear vesicle having been entirely removed. Drawing made three months after operation. Enlarged
skin on right side just dorsal to ear vesicle, and needle passed
diameters. ,
down between the neural tube and ear vesicle and moved backward and forward so as to sever its nervous connection without
otherwise disturbing the ear vesicle or loosening it from the skin.  
None of the four specimens studied swimmed properly, though
one of them could swim somewhat, but was easily confused by
any excitement and then made wild and ill directed movements.  
It was thought that the ear vesicles in these cases would escape
injury; but examination of the specimens when cut in serial sections




The operation was one that could be performed without difficulty. A tadpole about 3 mm. long is selected and the ear
Development of Amphibian Ear Vesicle
vesicle taken out on one side in the manner described above. The
specimen is then turned over and the opposite ear vesicle is
uncovered and loosened from the epidermis. Before actually
removing it a straight incision is made with scissors or needles in
front of the eye and a pocket is created by gently spreading the subjacent mesoderm apart until the brain is exposed. The loosened
ear vesicle is then lifted from its natural place and slipped into
this pocket. If the incision is carefully made the edges of the
wound close at once and on the following day there is no trace of
the operation left. Nine operations of this kind were made and
seven of the tadpoles successfully reared. While they were growing it could be seen from a surface view that the transplanted
vesicle was developing and causing a corresponding elevation in




443




showed that they were not perfectly normal. This experiment
might be repeated on a larger number of specimens and still
greater care used in severing the nerve connection, in which case
a perfect labyrinth could doubtless be obtained.


h (Rana catesbiana) Left ear vesicle transplanted into another specimen, in a subdermal pocket in the region of the prootic ganglion between the right eye and ear vesicle, thus the host
had three ear vesicles, two being on the right side. Twelve days
after the operation three out of four specimens so treated exhibited incoordinate movements. Here we have to consider the
crowding out of position of the normal right ear vesicle by the one
transplanted near it.


i Left ear vesicle removed; fine needle passed through the skin
so as to make a small puncture in the right ear vesicle; on withdrawal of the needle the edges of the wound immediately close and
there is no loss of cells from underneath or from the skin itself.
Of four specimens at the end of one month three were able to swim,
and this demonstrated the functional ability of an ear vesicle thus
treated.


j Left ear vesicle removed; small section of the covering skin
removed so as to expose the right ear vesicle, but otherwise it is
not disturbed and the nerve ganglion connection is left intact.
Five specimens were kept under observation for one month, and
four of them behaved throughout like those possessing one
untouched normal ear vesicle; except for slight incoordination
brought out by excitement they could swim properly.


On bringing together the results of these experiments, it becomes
immediately apparent that almost any operative procedure carried out on young larvae in the region of the ear vesicle seriously
interferes with the development of the function of that organ. It
is possible to lift a skin flap and expose it, and to make a needle
puncture in it without destroying its subsequent usefulness; but
any operation involving a loss of part of its wall or disturbing its
position and nerve-connection with the brain causes apparently
complete loss of function. The functional disturbance is out of all
proportion to the histological condition. There may be a labyrinth that to all appearances is perfectly formed and that seems to




555
444




Experiments on the Developing Ear Vesicle
George L. Streeter , M.D.


front of the eye. A sketch of one of these at the end of the third
month is shown in Fig. 6.


Their behavior during the first week following the operation was
have a normal nerve ganglion connection with the brain at the  
identical with that of specimens deprived of both vesicles as was
proper place, and yet the specimen may not have given signs of  
to be expected. Toward the fifth and sixth days they could
any functional activity on the part of that organ.  
make progress while touching the side or bottom of the dish, but
any attempt at swimming in free water resulted only in irregular
flexions of the body and somersaults. It was hoped that the transplanted vesicle might then begin to function and make it possible
for them to perceive their position while in free water, but this did
not occur. They continued to behave in all respects like tadpoles
having no labyrinth and never gave evidence of possessing any
trace of equilibrium. *


At the end of the third, fourth and twelfth weeks specimens
Spemann 6 is doubtless mistaken in attributing the disturbance
were killed in preserving fluid and prepared in serial sections.
in equilibrium simply to the alteration in the planes of the canals.
Examination of the sections showed that in six out of the seven
He reports some experiments in which at an early stage a skin flap
specimens which were cut, the transplanted vesicle had developed
was turned back, and the ear vesicle taken out and replaced in
to a greater or less extent, and it was these vesicles that formed the  
various positions; and in such specimens he observed faulty equilibrium, and on sectioning his material the vesicle seemed to lie in  
surface elevations that had been macroscopically visible in front
an abnormal position, and this he assumes to be the cause of the  
of the tadpole’s eyes. Graphic reconstructions of them are represented in Figs. 7 to I2. It will be seen that none of these constitute a perfect labyrinth, but on closer study it is found that they  
abnormal movements observed. On the one hand, wax plate
all possess certain features which are characteristic of it. In the  
reconstructions of misplaced ear vesicles show that in my cases they  
first place, that which was transplanted in the form of an open
regain their proper position, and the canals eventually lie in their
auditory cup developed after the operation into a closed vesicle
normal planes; the specimens nevertheless continue to make incoordinate movements. On the other hand, in those experiments
containing endolymph. This did not then remain a simple vesicle,  
where the normal position of the vesicle, as regards the planes
but exhibited the tendency to subdivision into two or more compartments, the utriculus and sacculus, as seen in Figs. 7,8,12. In the  
of space, was undisturbed the results were equally serious. My
walls ofthese compartments there are areas of specialized epithelium
own experiments suggest that the difficulty lies not so much with
representing the maculae acusticae. In Fig. 7 there opens out of
the end organ as with the central connections, and perhaps further
the more dorsal compartment a distinct endolymphatic appendage.
experiments in that direction would furnish additional information upon this subject.  
A typical semicircular canal is not present in any of them; but
what may be called a canal tendency is seen in Fig. 8, where
there is a tube uniting the two principal compartments. The
small blind pouches leading off the main vestibule, three of which
are present in Fig. 10, doubtless represent abortive canals. In
transverse section they are perfectly round and look like typical




556
CONCLUSIONS


The primitive ear vesicle of the tadpole may be loosened from
its normal position and rotated in various directions, so that its
axes lie in abnormal planes, and notwithstanding such interference it eventually develops into a labyrinth which is right side up
and exhibits the normal relations to the brain and the surrounding
structures. When transplanted to the opposite side of the body,
if placed in the acoustic region, it likewise assumes a normal
posture. Judging from these facts, the posture of the labyrinth
is controlled by its environment.


George L. Streeter
The “ laterality’’ of the labyrinth is determined before the closure of the ear vesicle. When the left ear vesicle is transplanted




canals. It may be recalled that Riidinger (’88) described the
6 Spemann, H., ’06: Ueber embryonale Transplantation. Verhandl. der Gesell. Deutscher Naturf.  
semicircular canals as developing in the form of blind tubes sprouting out from the general vesicle. It is quite possible that he was
u. Aerzte. 78 Vers. Stuttgart.  
dealingwith an abnormal embryo and had the same form of canals
that we see in Fig. lo.  


The ear vesicles are more or less completely enveloped in connective tissue membranes and they are partly incorporated in
masses of cartilage, some of which belongs to the normal cartilaginous cranium and some of it is the regular cartilaginous capsule
of the labyrinth, the two fusing together in some places.


In four cases (Figs. 7, 8, 9 and 10) a group of ganglion cells and
Development of Amphibian Ear Vesicle
nerve fibers are attached to the median side of the vesicle near its
caudal end and extend toward the central nervous system. In
one instance (Fig. 7) the nervous connection between vesicle and
brain at the junction of olfactory lobe and fore-brain, is complete,
though it is only a few fibers that actually enter the brain. As the
acoustic ganglion at the time of the operation is attached to the
auditory cup some of its ganglion cells are undoubtedly carried
along with it, and it is probable that it is these cells that furnished
the nerve connections just described. At the time the transplanted
ear cup was slipped into its pocket the adherent ganglion cells
must have been lodged in various positions as regards the ear cup
and the fact that they all come finally to lie on the median side of
the vesicle and lead toward the brain must be explained by some
theory of an attraction existing between brain and nerve.


When we have to deal with a transplanted labyrinth that has
reached a development equal to those that function in young tadpoles, and has established communication with the central nervous system, we might expect that it would show some sign of
physiological activity. The failure of it to do so is perhaps best
accounted for by the fact that the point of entrance into the brain
is so fa,r away from the hind-brain centers and the spinal cord that
connections with these are not established. If the experiments were
varied and the vesicle transplanted to some point in the neighborhood of the occipital nerves this difficulty would be obviated.


445


Experiments on the Developing Ear Vestcle


to the right side it retains its characteristics as a left-sided organ,
though it otherwise adapts itself to its new position in a normal
manner.


557
The functional disturbance, in experiments on the ear vesicle,
is out of all proportion to the histological appearances; any operation carried out in the acoustic region involving a loss of part of
the wall of the ear vesicle, or disturbing its position, or nerve connection with the brain results in faulty equilibrium; absence of
function was observed in cases where the labyrinth and its nerve
connections seemed to have attained perfect histological development.






Figs. 7 to 12. Graphic reconstructions showing the form and relations developed by transplanted ear
vesicles, one to three months after the operation. In all six cases the right ear vesicle was removed and
the left vesicle transplanted into a subdermal pocket between eye and nostril. In Figs. 7, 8, 9 and 10
the acoustic nerve and ganglion extended from ear vesicle toward brain; in Fig. 7 the connection was
complete, the fibers entering at junction of fore-brain and olfactory lobe. Central nervous system,
shaded; ear vesicle, solid black.






£ yj- ^


•V..v , * '




fi M.


Wt" ;




Line 681: Line 649:




r ,




?




Line 694: Line 664:




558




George L. Streeter
Conclusions




, In the tadpole the ear vesicles are essential for the development
of the power of equilibration, but the study of normal specimens
shows that well developed equilibration may be present before
the completion of the semicircular canals; the latter as such are
therefore not essential.


When both vesicles are removed no other organ compensates
for their loss and the animal is completely and permanently helpless as regards the maintenance of equilibrium. When only one
ear vesicle is taken out the remaining vesicle is capable of performing the work of both so perfectly, that the casual observer would
mistake them for normal individuals.


Transplantation of the ear vesicle shows that the group of cells
forming the auditory cup or primitive ear vesicle is specialized to
that degree that although removed from their natural relations
and placed in a new environment they still continue to differentiate
themselves into a structure approximating the normal labyrinth.
A nerve and ganglion develops, and complete nervous connection
may be established between the transplanted vesicle and the brain
at an abnormal place. Where the latter occurred it did not give
evidence of any functional ability.


 
. 1. 1
LITERATURE
 
Lee, F. S., ’93.—A Study of the Sense of Equilibrium in Fishes. Journ. of Physiol.,
vol. XV and xvii.
 
’98.—The Function of the Ear and Lateral Line in Fishes. Amer. Journ.
 
. of Physiol., vol. i.
 
Lewis, W. H., ’04.—Experimental Studies on the Development of the Eye in
Amphibia. Amer. Journ. Anat., vol. iii.
 
Lyon, E. P., ’00.—A Contribution to the Comparative Physiology of Compensatory
Motions. Amer. Journ. of Physiol., vol. iii.
 
Parker, G. H., ’05.—^The Skin, Lateral-Line Organs and Ear as Organs of
Equilibration. Proceedings of Amer. Zool. Soc., Science, vol. xxi.
 
Prentiss, C. W., "01.—The Otocyst of Decapod Crustacea; its Structure, Development and Functions. Bull. Museum Compar. Zool., Harvard
College, vol. xxxvi.
 
Rudinger, ’08.—Zur Anatomie u. Entwickelungdes inneren Ohres. Berlin, 1888.  




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Latest revision as of 11:48, 9 January 2020

https://archive.org/details/somefactorsindev00stre/page/432

AMPHIBIAN EAR VESICLE AND FURTHER EXPERIMENTS ON EQUILIBRATION


B y

George L. Streeter, M D.


REPRINTED FROM

THE JOURNAL OF EXPERIMENTAL ZOOLOGY

Volume IV No. 3


BALTIMORE, MD., U.S. A. September, 1907




ii M\ ^ ^ IXC '


From the Wistar Institute of Anatomy and Biology, Philadelphia


SOME FACTORS IN THE DEVELOPMENT OF THE AMPHIBIAN EAR VESICLE AND FURTHER EXPERIMENTS ON EQUILIBRATION

i KY

GEORGE L. STREETER, M.D.

Associate Professor of Neurology at the Wistar Institute With Six Figures

In a previous paper concerning experiments on the developing ear vesicle 1 it was shown that the group of cells forming the primitive epithelial ear cup or ear vesicle of the tadpole is specialized to that degree that although removed to an abnormal environment the cells still continue to differentiate themselves into a structure possessing many of the features of a normal labyrinth. Recently it has been shown by Lewis 2 that even earlier, while still an uninvaginated plate, the ear anlage is already capable of a certain degree of independent differentiation. In the following paper additional evidence will be given of the high degree of developmental independence possessed by the early labyrinth cells. It will be pointed out that individual parts of the vesicle may develop independently of the rest of the vesicle. It will also be shown that the process of differentiation extends to the difference existing between a right and left-sided organ. A left ear vesicle transplanted into the empty pocket left by the removal of the right ear vesicle develops into a labyrinth that is perfect in general form and in its relations to the brain, with the exception that it maintains its left-sided character; the anterior semicircular canal is found on the caudal side toward the vagus group, while the posterior canal lies toward the eye, and likewise the lagena which

1 Streeter, G. L., *o6: Some experiments on the developing ear vesicle of the tadpole with relation to equilibration. Jour, of Experimental Zool., vol. iii.

2 Lewis, W. H., ’07: On the origin and differentiation of the otic vesicle in amphibian embryos. Anatomical Record, No. 6, Amer. Jour, of Anat., vol. vii.

The Journal of Experimental Zoology, vol. iv, no. 3.

(5 1 & 3


43 2


George L. Streeter , M.D.


normally buds out from the caudal border of the saccule in these cases is found extending forward toward the prootic ganglion.

The ear vesicle, however, is not in all respects independent of the surrounding structures. Some experiments which are reported below, indicate that its position in reference to the brain, ganglion masses and the surface of the body is determined by the environment itself; it may be rotated in any direction, and nevertheless it eventually develops in the normal attitude, with the saccule toward the ventral surface, the semicircular canals toward the dorsal surface, the lateral semicircular canal being toward the lateral surface, and the endolymphatic appendage toward the brain.

The experiments were carried out on larvae of Rana sylvatica and Rana pipiens, and the operating stage was the same that was used in previous experiments. 3 The time is just at the close of the non-motile stage, and the epithelial ear consists of an invaginated cup-shaped mass of cells just in the process of being pinched off from the deeper layer of the skin, with the edges turning in to form a closed vesicle. For simplicity the term “ear vesicle” will be used even though the closure is not yet complete; the attempt to distinguish between auditory cup and auditory vesicle does not seem to be justified for the present purposes. The technique of the operations was also the same as that described in the previous paper. Notes were made on the behavior of the animals, and at the end of from four to six weeks the specimens were preserved in a chrome-acetic mixture, cut in serial sections, and stained with haematoxylin and congo red. With certain specimens the ear vesicle, adjacent ganglia, and a portion of the central nervous system were reconstructed after the Born wax plate method. Eleven such models were made, and photographs of some of them are reproduced in Figs. 2, 3 and 6. With the aid of these models it was possible to identify relations and detailed features of the labyrinths that otherwise could not have been recognized.

The morphological features of the experiments will be first considered, and the behavior of the animals and its relation to equilibrium will be treated separately in the latter part of the paper.


Streeter ’06: /. c., Fig. 3, p. 547.


Development of Amphibian Ear Vesicle


433


DETERMINATION OF POSITION OF THE EAR VESICLE

The conclusion that the attitude of the developed labyrinth, the position of its canals and various chambers, is determined by its environment is based on seventeen experiments in which the ear vesicle was loosened from its normal situation and placed in an abnormal attitude, and the specimen then allowed to continue in its development. At the end of a month examination showed that the labyrinth had become differentiated with varying degrees of completeness, and in each instance had developed in normal relation to the surrounding structures.

Rotation in Two Directions. In eight of these experiments the ear vesicle was rotated i8o° around both its vertical and transverse axes, so that it was turned face inward and upside down ; or, in other words, its lateral or invaginated surface was toward the brain and its ventral border was where the dorsal border should be, the maximum displacement. After this procedure the wounds healed within a few hours, and the larvae were reared up to the fourth or fifth week, when they were killed and cut in serial sections. The labyrinths of five specimens were reconstructed. Before describing them reference should be made to the normal condition of the labyrinth at this age. A reconstruction of a normal one with its adjacent structures is shown in Fig. I.

From the reconstruction of a normal specimen it can be seen that the three semicircular canals have individual characteristics by which they can be separately identified; such as the Y-shaped union of the anterior and lateral canals, and the overlapping of the caudal end of the lateral canal by the posterior canal, and the junction of the posterior and anterior canals to form the crus commune. The differentiation between utricle and saccule is not yet complete, but the part that is to become saccule is so labeled. From the caudal border of the saccule can be seen a small pocket budding out which constitutes the lagena or primitive cochlea. Directly median to the crus commune is the endolymphatic appendage, consisting of a small duct leading from the main labyrinth chamber up between the labyrinth and brain to a rounded pouch, the saccus endolymphaticus. In their histology, as well as in


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their general form, the various parts of the labyrinth exhibit at this time individuality. (See Fig. 4.) The ventro-median portion of the vestibular sac and the ampullar ends of the semicircular canals possess high columnar cells forming the neuro-epithelial maculae which are supplied with fibers from the acoustic ganglion, lying against the medial wall of the labyrinth. The endolymphatic sac has cuboidal cells, and the lagena has intensely staining columnar cells like those seen in the macular regions. The lagena is further characterized by its sharply rounded outline, and by the fact of its being compactly surrounded by ganglion cells and fibers, and cartilage forming cells. These features are so definite that

Crus commune


Fig. 1 Reconstruction showing the form and relations of the membranous labyrinth of a normal tadpole (Rana pipiens) one month old. The labyrinth, adjacent ganglia and part of the brain w.. re reconstructed after the Born method, and the remainder of the figure was drawn from a dissection of a tadpole of the same age. Enlarged 35 diameters.

the various parts of the labyrinth can be recognized without difficulty, even though they happen to be incomplete, or out of their normal relations.

Now if one examines the models of the operated specimens, photographs of three of which are reproduced in Fig. 2, it is seen that the individuality of the semicircular canals can at once be identified. In model a , the canals are practically normal; in model b, the anterior canal is small, and the lateral canal consists only of a pouch which has not been pinched off from the main cavi-ty; in model c , the posterior canal remains a simple pouch, while the


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anterior and lateral canals are normal. In considering the posture of the canals it is to be noted that the surrounding structures have been left out in Fig. 2, to avoid unnecessary duplication; the three models are all represented in the same relative position as that of the labyrinth in Fig. 1, 1. e., the cephalic end is on the right, the caudal end is on the left, the ventral surface is below, and the dorsal surface is above. Thus it will be seen that the lateral canals in all three models are in the same plane; likewise the posterior canals all form the dorso-caudal border of the labyrinth, and the anterior canals form the dorso-cephalic border. The fact that the anterior canal is small in model b , 4 and the posterior canal is small in model c , gives rise to a false impression of a backward


sac. endolymph.


laeena

a h b C


Fig. 2 Reconstructions showing the form and posture developed by three labyrinths one month old’ which while primitive ear vesicles were rotated from their normal position so as to lie face inward and upside down. The models are placed so that their planes are parallel with those in Fig. I. Thus they present a lateral view with the cephalic end toward the right, caudal end toward the left, dorsal surface above, and ventral surface below. Enlarged 50 diameters.

and forward tilting of the vesicle. The saccule and lagena have the same position as in Fig. i, and the lagena points caudally as it should do. The endolymphatic appendage lies on the median side of the crus commune; in models b and c it is small, but the tip

4 This may be due to injury received at the time of operation. Such localized defects are frequently seen. They may involve any part of the labyrinth, and they vary greatly in the extent of the labyrinth wall affected. In one case the entire labyrinth was defective, with the exception of the endolymphatic appendage, which was normal in structure and position, and presented a curious appearance, being attached to the small irregular vesicle representing the labyrinth. Such localization of abnormal development is evidence of the high degree of specialization of the cells forming the primitive ear vesicle.


43 6


George L. Streeter , M.D.


of it can be seen in model a. The acoustic nerve and ganglion are attached to the median and ventral surfaces of the labyrinth, and the nerve connection with the brain appears to be normal.

The conditions found in the three specimens pictured in Fig. 2 are typical of what is found in the other five specimens examined. They vary in the completeness of their differentiation, some of them consisting of only a vesicle with perhaps a single canal pouch, but in all cases the acoustic ganglion is present on the ventro medial surface, and the macular areas can be recognized. The lagena is present in seven out of eight cases. The endolymphatic appendage developed in six out of eight cases. As regards posture, the rule is that the more perfectly the labyrinth is developed the more accurately its posture corresponds to the normal relations. But even in the most imperfect specimens when the endolymphatic appendage appears it is on the medial surface, and the tendency to canal formation is always on the dorso-lateral surface, and the saccule and lagena appear on the ventral surface. This condition of course applies only to vesicles that have been implanted in the acoustic region as was done in all the above cases.

Rotation in One Direction. In four experiments the ear vesicle was rotated 180° around its vertical axis, i.e> turned face inward. These specimens were then reared as in the preceding instance, and eventually cut in serial sections. A reconstruction model of one of them is reproduced in Fig. 3, and if it is compared with Fig. 1 it will be seen that although the vesicle was started in its development with invaginated side toward the brain yet the completed labyrinth has the normal posture. A section of the same specimen is reproduced in Fig. 4, showing the labyrinth surrounded by developing cartilage. The acoustic ganglion is connected in normal manner with the brain and sends peripheral fibers to the thickened floor of the saccule. The endolymphatic sac is in its normal position, and the narrow duct can be seen connecting it with the main chamber of the labyrinth directly median to the crus commune. The series through this specimen show that histologically it is practically perfect. Of the other three specimens one was almost equally perfect, another showed some abnormalities in the formation of the canals and the lagena, and the


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fourth was quite imperfect, consisting of only a large vesicle with a thickened epithelial floor connected by a few nerve cells and fibers with the brain.


Fig. 3 Reconstruction of a tadpole labyrinth one month old, which when a primitive ear vesicle was rotated from the normal position i8o° in one direction, so as to lie with invaginated side toward the brain. A section through the same labyrinth is shown in Fig. 4. Enlarged 55 diameters.


Fig. 4 Section through the membranous labyrinth shown in Fig. 3. It shows that though originally turned face inward it has developed in the normal attitude, e, endolymphatic appendage; c.c., crus commune; sacc., saccule; c. lat., lateral semicircular canal; gang, acust., acoustic ganglion. Enlarged 55 diameters.

Transplanted Specimens. The irregularity of form of the six specimens transplanted to the region between the eye and nostril, previously reported , 5 is so great that they give no assistance in solv

Streeter ’06, l. c., p. 557.



43 8


George L. Streeter , M.D.


ing the question of posture. However, in five cases, which will be presently described, where the ear vesicle was transplanted from the left side to the right side into the place made vacant by the removal of the right ear vesicle, in spite of the fact that these ear vesicles were implanted with haphazard attitude toward the adjacent structures, they nevertheless in each instance, developed right-side up, and with the median surface toward the brain, as can be seen in Figs. 5 and 6.


Mesen

Sac.ehdolymph j


ce p h. Dience p h.


Fig. 5 Reconstruction showing the form and relations developed by a left ear vesicle when transplanted to the right side; it shows that under such circumstances the ear vesicle retains its left-sided characteristics, though it otherwise normally adapts itself to its new situation. A photograph of the same specimen is shown in Fig. 6, c.


DETERMINATION OF THE D EXTRAL AND SINISTRAL CHARACTER OF THE EAR VESICLE

The question as to whether the right or left-sidedness of the ear labyrinth is controlled by the environment, or is determined by some intrinsic character of its own constituent cells, is answered in favor of the latter by the fact that if the left primitive ear vesicle, before the time of its complete closure, is transplanted to the opposite side of the embryo it retains its original left-sidedness. In five specimens, at the usual operating stage, the right ear vesicle was removed, and at the same time the left ear vesicle was uncovered and lifted from its natural bed and then placed into the pocket


Development of Amphibian Ear Vesicle


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from which the right vesicle had been taken and allowed to heal. In making the transplantation no effort was made to place the ear vesicles in any particular posture. After keeping the specimens alive for one month they were sectioned and from three of them reconstructions were made of the transplanted ear vesicle together with the adjacent structures. The three labyrinths are shown in Fig. 6, and model c is again shown in Fig. 5, with the brain included. It will be seen that in developing they have assumed the normal attitude toward the brain. The endolym

sac. endolymph.


lagena

a be


Fig. 6 Reconstructions of three labyrinths which while primitive ear vesicles were transplanted from the left to the right side. They are all represented in the same position as the models in Fig. 2. The model c is the same that is shown in Fig. 5. Enlarged 50 diameters.

phatic appendage and the median side of the labyrinth is toward the brain, the semicircular canals are toward the dorso-lateral surface, and the saccule and lagena are toward the ventral surface. But it can at once be recognized that the saccule and lagena point forward toward the eye, and that the anterior and posterior canals are in reversed positions. We thus have a complete mirror image of the right labyrinth, i. e a left labyrinth. Model a possesses three semicircular canals and is almost a normally formed left labyrinth. In model b the lateral canal consists of a pouch whose walls did not undergo the customary approximation and central absorption. In model c the posterior canal is not pinched off. In each of the models the lagena, saccule, and endolymphatic


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George L. Streeter , M.D.


appendage are typical, and there is establishment of normal appearing nerve and ganglion connections.

EQUILIBRATION

It was found in the experiments performed a year ago that removal of one or both ear vesicles, just after they are pinched off from the skin, produces in the tadpoles definite disturbances in the development of their power of equilibration. It was found that when a tadpole is deprived of but one ear vesicle he is by virtue of the remaining one able to develop practically normal swimming abilities; but when both ear vesicles are removed the results are more serious, and in that case the tadpole never develops any sense of equilibrium and is never able to swim. The loss is not compensated for by any other organ and the animal lies helpless on the bottom of the dish. With one ear vesicle the tadpole swims practically in normal fashion, and with no ear vesicle he cannot swim at all.

The fact that one ear vesicle is sufficient for the maintenance of equilibrium greatly simplifies the study of this mechanism; it means that one side can be immediately eliminated, and the problem is reduced from a bilateral one to a unilateral one. A series of experiments at once suggested themselves, in which the ear vesicle of one side was to be removed, and then various operative procedures undertaken upon the ear vesicle of the opposite side, and the test of its consequent functional ability was to be the very decisive one of whether the animal could swim properly, or whether it could not swim at all.

In the paper referred to there is described the experiment of transplanting the ear vesicle into a subdermal pocket in from of the eye. When this was done the transplanted ear vesicle continued in its development, and in some instances established a nerve-ganglion connection with the forebrain; but such specimens never gave evidence of functional activity. The failure to functionate was not unexpected, inasmuch as the connections established were at an abnormal situation, and furthermore the vesicles though having developed many essential features of the normal labyrinth


Development of Amphibian Ear Vesicle


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were still quite imperfect in the formation of the separate chambers and the semicircular canals. So this year in carrying out the experiments described in the first part of the present paper the behavior of the specimens was eagerly watched, and the endeavor was made to determine the amount of alteration in position and defectiveness in form that is compatible with functional activity, involving the problem of the correlation between function and morphology. The observations made in the different experiments have been arranged and condensed as follows:

a Left ear vesicle removed; right ear vesicle loosened from skin and rotated, in six specimens around the vertical axis 180° and in eight specimens around both the vertical and transverse axis 180 0 . As has already been shown these ear vesicles developed into labyrinths of varying degrees of perfection, some being completely normal in form and having apparently normal ganglion and nerve connection with the brain wall. (See Figs. 2, 3 and 4.) The behavior of all the specimens was uniform, both where the ear vesicle was rotated in one plane and where rotated in two planes; at the end of a week after the operation, when with a normally functionating labyrinth they should be able to swim freely and directly, they instead exhibit only irregular movements or spin around in spirals or circles. Their incoordinate movements continue, and at the end of a month there is no improvement; i.e 9 they behave exactly like specimens with both ear vesicles removed. Evidently ear vesicles thus treated do not perform their natural function.

b Left ear vesicle removed; right ear vesicle fragmented by teasing between the points of two needles, the fragments left in place. Ten specimens were treated in this way, and were kept under observation four weeks, during which time they gave no evidence of any sense of equilibrium.

c Right ear vesicle removed; left ear vesicle transplanted to the empty pocket on the right side. Five specimens were operated upon and observed for one month, at the end of which time they were cut in serial sections, and it was found that the ear vesicles had developed into fairly complete labyrinths, but had maintained the characteristics of a left-sided organ. (Figs. 5 and 6.)


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George L. Streeter , M.D.


Throughout the whole period of observation they had exhibited incoordinate movements, and at the end of that time they were unable to swim. This and the two previous operations indicated that rotation of an ear vesicle, or transplanting it from one side to the other, or fragmenting it was not compatible with the development of its function, in spite of the fact that the ear vesicle proceeded in its development and had become to all appearances almost a perfect labyrinth. In the next experiments less severe treatment was tried.

d Left ear vesicle removed; right ear vesicle uncovered and carefully lifted out and then immediately placed back in its original position, the effort being made to do a minimum amount of injury. Of six specimens all exhibited symptoms of the absence of all sense of equilibrium.

In the experiments a, b , c and d there was the possibility of injury to both the nerve-ganglion connection and the ear vesicle. In the following experiments the effort was made to restrict the injury to one or the other. 1

e Left ear vesicle removed; right ear vesicle uncovered and a fragment cut from the cephalic portion of its wall, care being used not to otherwise disturb the vesicle. Eight such specimens were kept five weeks, and none of them developed any sense of equilibrium, or were able to swim.

f Left ear vesicle removed; right ear vesicle uncovered and a small piece cut from its caudal border, any further disturbance being avoided as in e. Eight specimens were operated upon, and after keeping them four weeks none of them could swim properly.

g Left ear vesicle removed; longitudinal incision made through skin on right side just dorsal to ear vesicle, and needle passed down between the neural tube and ear vesicle and moved backward and forward so as to sever its nervous connection without otherwise disturbing the ear vesicle or loosening it from the skin. None of the four specimens studied swimmed properly, though one of them could swim somewhat, but was easily confused by any excitement and then made wild and ill directed movements. It was thought that the ear vesicles in these cases would escape injury; but examination of the specimens when cut in serial sections


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showed that they were not perfectly normal. This experiment might be repeated on a larger number of specimens and still greater care used in severing the nerve connection, in which case a perfect labyrinth could doubtless be obtained.

h (Rana catesbiana) Left ear vesicle transplanted into another specimen, in a subdermal pocket in the region of the prootic ganglion between the right eye and ear vesicle, thus the host had three ear vesicles, two being on the right side. Twelve days after the operation three out of four specimens so treated exhibited incoordinate movements. Here we have to consider the crowding out of position of the normal right ear vesicle by the one transplanted near it.

i Left ear vesicle removed; fine needle passed through the skin so as to make a small puncture in the right ear vesicle; on withdrawal of the needle the edges of the wound immediately close and there is no loss of cells from underneath or from the skin itself. Of four specimens at the end of one month three were able to swim, and this demonstrated the functional ability of an ear vesicle thus treated.

j Left ear vesicle removed; small section of the covering skin removed so as to expose the right ear vesicle, but otherwise it is not disturbed and the nerve ganglion connection is left intact. Five specimens were kept under observation for one month, and four of them behaved throughout like those possessing one untouched normal ear vesicle; except for slight incoordination brought out by excitement they could swim properly.

On bringing together the results of these experiments, it becomes immediately apparent that almost any operative procedure carried out on young larvae in the region of the ear vesicle seriously interferes with the development of the function of that organ. It is possible to lift a skin flap and expose it, and to make a needle puncture in it without destroying its subsequent usefulness; but any operation involving a loss of part of its wall or disturbing its position and nerve-connection with the brain causes apparently complete loss of function. The functional disturbance is out of all proportion to the histological condition. There may be a labyrinth that to all appearances is perfectly formed and that seems to


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George L. Streeter , M.D.


have a normal nerve ganglion connection with the brain at the proper place, and yet the specimen may not have given signs of any functional activity on the part of that organ.

Spemann 6 is doubtless mistaken in attributing the disturbance in equilibrium simply to the alteration in the planes of the canals. He reports some experiments in which at an early stage a skin flap was turned back, and the ear vesicle taken out and replaced in various positions; and in such specimens he observed faulty equilibrium, and on sectioning his material the vesicle seemed to lie in an abnormal position, and this he assumes to be the cause of the abnormal movements observed. On the one hand, wax plate reconstructions of misplaced ear vesicles show that in my cases they regain their proper position, and the canals eventually lie in their normal planes; the specimens nevertheless continue to make incoordinate movements. On the other hand, in those experiments where the normal position of the vesicle, as regards the planes of space, was undisturbed the results were equally serious. My own experiments suggest that the difficulty lies not so much with the end organ as with the central connections, and perhaps further experiments in that direction would furnish additional information upon this subject.


CONCLUSIONS

The primitive ear vesicle of the tadpole may be loosened from its normal position and rotated in various directions, so that its axes lie in abnormal planes, and notwithstanding such interference it eventually develops into a labyrinth which is right side up and exhibits the normal relations to the brain and the surrounding structures. When transplanted to the opposite side of the body, if placed in the acoustic region, it likewise assumes a normal posture. Judging from these facts, the posture of the labyrinth is controlled by its environment.

The “ laterality’’ of the labyrinth is determined before the closure of the ear vesicle. When the left ear vesicle is transplanted


6 Spemann, H., ’06: Ueber embryonale Transplantation. Verhandl. der Gesell. Deutscher Naturf. u. Aerzte. 78 Vers. Stuttgart.


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to the right side it retains its characteristics as a left-sided organ, though it otherwise adapts itself to its new position in a normal manner.

The functional disturbance, in experiments on the ear vesicle, is out of all proportion to the histological appearances; any operation carried out in the acoustic region involving a loss of part of the wall of the ear vesicle, or disturbing its position, or nerve connection with the brain results in faulty equilibrium; absence of function was observed in cases where the labyrinth and its nerve connections seemed to have attained perfect histological development.




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