/ . Embryol. exp. Morph. Vol 34, 3, pp. 657-667, 1975
Printed in Great Britain
657
The effect of ablation of the olfactory pits
on the development of the habenular nuclei in
Rana pipiens
By BYRON K. NORRIS 1 AND VICTOR B. EICHLER 1
From the Department of Biology, Wichita State University, Kansas
SUMMARY
The habenular nuclei in the diencephalon of the frog, Rana pipiens, are asymmetrical
structures: two discrete cell groups develop on the left side (as medial and lateral nuclei),
while a single nucleus is formed on the right side. Experimental animals were subjected to
bilateral removal of the olfactory pits at an early embryonic stage, and were maintained with
normal control animals until metamorphosis was complete. The length, relative volume and
cell number for each of the three nuclei were determined in the control and experimental
animals at regular intervals during larval development. In the control animals, the left medial
nucleus developed similarly to the right nucleus spatially and temporally; however, the left
lateral nucleus was significantly different in its development in the three parameters measured.
In the experimental animals the left medial and the right habenular nuclei were alone affected
by the removal of the olfactory pits. The results provide experimental evidence that the right
and left medial, but not the left lateral, habenular nuclei are centers receiving afferent
olfactory fibers.
INTRODUCTION
The habenular nuclei in the brain of vertebrates are small clusters of cells
located in the anterior dorsal diencephalon, at the level of the epiphysis. The
nuclei appear as elongated oval structures with compact neural cell bodies
surrounding a relatively cell-free interior. In amniote vertebrates these groups
of nerve cell bodies are symmetrically located on either side of the third ventricle;
however, many anamniotes exhibit a left-right asymmetry.
The first report of the existence of this asymmetry appears to have been by
Goronowitsch (1833), who described the asymmetry in the habenula of the
sturgeon. In this species of fish, the right nucleus was found to be strikingly
larger than that of the left side. Gaupp (1896) published his findings on the
asymmetrical nature of the frog's habenulae, and showed that on the right side
there is a single nucleus, while the left side has a large medial nucleus and a
much smaller lateral nucleus. Within the next five years several reports appeared
indicating that a left-right asymmetry existed also in the habenular nuclei of a
variety of cyclostomes and bony fishes.
1
Authors' address: Department of Biology, Wichita State University, Wichita, Kansas
67208, U.S.A. Requests for reprints should be sent to Dr Eichler.
658
B. K. NORRIS AND V. B. EICHLER
However, the findings of an anatomical asymmetry in the vertebrate brain
were generally ignored during the first half of the present century, for it was
generally accepted that the vertebrate brain was composed of mirror image
halves. The existence of asymmetries in the brain of amphibians, reptiles, birds
and mammals was even specifically denied during this time in books authored
by such eminent investigators as Kappers, Huber & Crosby (1936) and Beccari
(1943).
Beginning in the early 1950s, at the time Frontera (1952) published his very
detailed account of the anuran diencephalon, there had been little attention
paid to the interesting asymmetry present, in its development or its fiber
connections. But in the last five years, several investigators have noted in
independent studies (Braitenberg & Kemali, 1970; Morgan, O'Donnell &
Oliver, 1971; Eichler & Norris, 1974) that in the frog brain there are indeed
two distinct cell groups on the left side, while in the corresponding position on
the right side there is only a single group of cells.
The present communication reports our findings relative to two interesting,
but unanswered questions. We have asked, first, what is the pattern of development of this asymmetry in anuran amphibians? That is, what comparisons can
be made temporally and spatially in the normal development of the cell group
on the right side with each of the two cell groups on the left side? Secondly,
we asked what the nature of the neural input is to these nuclei. We wondered if
functionally, do both of the nuclei on the left side have the same neural input
as that on the right side and, if not, which of the left nuclei is functionally
analogous to the right one ?
MATERIALS AND METHODS
Eggs were obtained from adult Rana pipiens females by induced ovulation
and subsequent fertilization in the laboratory following the method of Rugh
(1934). Five days post-fertilization the olfactory pits of the experimental frog
embryos were surgically extirpated using fine glass needles and hair loops, under
a dissecting microscope. Approximately one hundred and fifty embryos were
operated on in full-strength Holtfreter's solution, and they were kept in this
solution for 2-3 h following the operation in order to promote wound healing.
Groups of ten embryos were kept in glass finger bowls containing 10 % Holtfreter's solution for 1 or 2 post-operative days and then were transferred to
plastic dish pans (30 x 33 x 15 cm) which held two liters aerated tap water. All
animals were maintained in uncrowded condition at room temperature and
were exposed to approximately equal numbers of light and dark hours. On the
eleventh day washed, canned spinach was placed into the pans so food was
available ad libitum. Food and water were changed daily.
Ten control and ten experimental animals were sacrificed at each of the
following development stages of Taylor & Kollros (1946): stage I, V, X, XV,
Development of frog habenular nuclei
659
XX and XXV, which correspond roughly to ages of 10, 20, 40, 60, 80 and 100
days, respectively. At stage XXV the animals have completed metamorphosis:
all four limbs have emerged, the tail is completely resorbed, and the animal is
ready to emerge from the water onto land.
The heads of the younger animals (stages I and V) were removed from the
body and placed in Bouin's fixative for 2 days. The heads of the older larvae
were trimmed by removing the lower jaw, and only the dorsal part of the head
was placed in the fixative. The tissues were dehydrated in alcohols, cleared in
methyl salicylate followed by xylene, and embedded in paraffin. Serial sections,
cut at 10 /.im, were stained with Delafield's hematoxylin and counterstained
with eosin.
The length of each habenular nucleus was determined by counting the number
of sections containing each one, and this number was multiplied by the section
thickness. The relative volume of each nucleus was determined by the method
of 'paired comparisons', where an image of each section of the right, left
medial, and left lateral habenular nucleus was projected at a magnification of
x 165, the outline of the projected image was carefully traced onto bond paper,
and each tracing (representing one 10 /m\ thick section through one of the
habenular nuclei) was then cut out. The combined weights of the tracings
representing each of the three nuclei at the various developmental stages, when
compared, gave a measure of the relative volumes of the respective structures.
The total number of cells in each nucleus were determined by counting the
cells in ten representative non-adjacent sections in every animal except the two
youngest stages, where only the nuclei in five non-adjacent sections were
counted. The cells around the perimeter and contained within the lumen of each
nucleus were counted at a magnification of x 100 using a hand tally counter.
The average of cells contained in ten sections was multiplied by the total number
of sections in the specific nucleus to give an estimation of the total cell population. A correction for cell estimates was determined following the method of
Ebbesson & Tang (1965).
The statistical comparison of the length, relative volume, and total number
of cells present in each of the three habenular nuclei of the control and experimental animals is presented in Table 1.
RESULTS
A. Length of habenular nuclei
Fig. 1 compares the length of the right, left medial and left lateral nuclei.
In Fig. 1A it can be seen that the left lateral nucleus develops later than the left
medial nucleus; it is not present at either stages I or V. Also, the left medial
nucleus is at all stages significantly longer than the left lateral nucleus.
There is a dramatic increase in the length of the control and experimental left
medial nucleus during stages I through XV. At stages XV through XX there is
42
E M B 34
660
B. K. NORRIS AND V. B. EICHLER
Table 1. Comparisons of length, relative volume, and number of cells in
each of the habenular nuclei of control and experimental animals^
Larval
stage
I
Length /tm
58
Right c
e
58
Left
c
70
65
medial e
Left
c
0
0
lateral e
Relative volume
002
Right c
e
002
Left
c
004
medial e
004
Left
c
0
lateral e
0
Cell number
Right c 615
e 565
Left
c 491
medial e 452
Left
c
0
lateral e
0
V
X
97
95
116
110
0
0
313
329
338
351
98
109
013
010
015
010
0
0
922
840
950
1020
0
0
XV
415
443
433
465
176
179
0-20
018
0-28
0-24
009
006
3244
4451
3360
4235
562
712
0-68
0-56*
0-72
0-63
014
013
7906
8510
5592
9968
1294
1208
XX
XXV
535
480
499
486
190
184
550
4081
595
392f
182
176
0-74
0-60*
0-90
0-70*
019
018
13925
11161*
9928
8777
1249
1168
0-89
0-68**
0-95
0-71*
0-23
0-22
15162
10466**
11139
6869**
1249
1249
c, control animals;
e, experimental animals.
* P < 005, Student's /-test;
** P < 001, Student's Mest.
f Each value represents the mean score for ten animals.
Left
B
Right
— — Control
**°** Experimental
V X XV XX XXV
Larval stage
V X XV XX XXV
Larval stage
Fig. 1. Length of the habenular nuclei of control and experimental animals; A, left
medial and left lateral nuclei; B, right nucleus.
Development of frog habenular nuclei
661
a leveling off of the rate of development of the experimental left medial nucleus
compared to the control animals increase. At stages XX-XXV there is a significant decrease in the length of the experimental left medial nucleus while the
length of the control medial nucleus continues to increase.
In Fig. 1A the lengths of the left lateral nuclei in experimental and control
animals are also compared. Animals in both experimental and control groups
exhibit no left lateral nucleus at stages I or V. At all stages examined thereafter,
the left lateral nucleus was found to be present. Its development, however, is
significantly reduced in comparison to the medial nucleus of the left side. The
length of the left lateral nucleus of the experimental animals is not significantly
different from the length of the left lateral nucleus of the control animals at any
of the stages examined. The length of the control and experimental left lateral
nucleus increases between stages V and XV. At stages XV through XXV the
length of the left lateral nucleus in larvae of both the experimental and control
groups neither increases nor decreases significantly as is observed in the left
medial nucleus.
In Fig. IB the length of the right nucleus in the experimental and control
animals is compared. The length of the control and experimental right nucleus
greatly increases between stages I through XV. Between stages XV to XX there
is a leveling off of the rate of development of the experimental right nucleus in
terms of length as compared to the continued increase in length of the control
right nucleus. Between stages XX and XXV in the experimental group of larvae
there is a significant decrease in the length of the right nucleus comparable to
that found in the left medial nucleus of the same animals, while the right nucleus
in the control animals increases in length throughout development similar to
the medial nucleus on the left side of these animals.
A similarity clearly exists in the development of the left medial and right
nuclei of larvae in both the control and experimental groups. Also, only the left
medial and the right nuclei are affected by the ablation of the olfactory pits as
shown by the decrease in the length of these cell groups in later developmental
stages. The development of the lateral nucleus begins later, it develops slower
and remains smaller than the other two habenular nuclei; it also is not significantly affected by extirpation of the olfactory pits at any of the larval stages
studied. These features clearly set the left lateral nucleus apart from the left
medial and the right nucleus.
B. Relative volume of habenular nuclei
The relative volumes of each of the three nuclei were determined at the six
selected larval stages in both the experimental and control groups. The results
are compared in Fig. 2 A for the left medial and the left lateral nuclei. The
relative volumes of the left medial nuclei of animals in the control and experimental groups show a great increase between stages I through XV. The
relative volume of the left medial nucleus is less at all stages examined in the
42-2
662
B. K. NORRIS AND V. B. EICHLER
experimental animals when compared to the unoperated ones, but the difference
is only of statistical significance at stages XX and XXV, the same stages where
the decrease in the length was observed. The relative volume of the left lateral
nucleus of the animals in the experimental and control groups is not significantly
different at any larval stage examined, but at every stage examined the lateral
nucleus is significantly smaller than the medial nucleus.
Right
V
X
XV XX XXV
Larval stage
V
X
XV XX XXV
Larval stage
Fig. 2. Relative volume of the habenular nuclei of control and experimental
animals: A, left medial and left lateral nuclei; B, right nucleus.
Fig. 2B compares the relative volumes of the right nucleus in control and
experimental animals. The relative volume of this nucleus increases in both
groups between stages I and XV. The volume difference in this nucleus between
the two groups becomes significant by larval stage XV. This reduction in volume
reflects the decrease in the length observed in the right nucleus of the experimental animals. The development of the left medial and the right nucleus in
larvae of both the experimental and control groups in terms of volume, shown
in Fig. 2, indicates that the left medial and the right nuclei are not significantly
different, while a significant difference in this parameter is noted between the
left lateral and right nuclei through all of the larval period.
C. Number of cells in the habenular nuclei
In Fig. 3 the total cell population of all three nuclei are shown. Fig. 3 A
compares the total cell population for the two left nuclei in both the experimental
and control animals. The left medial nucleus contains significantly more cells at
all stages examined than does the left lateral nucleus in both the control and
Development of frog habenular nuclei
663
experimental groups. The number of cells contained in the left medial nucleus
increases between stages I through XV in animals in both the experimental and
control groups. There is less increase in the number of cells contained in the left
medial nucleus of the experimental animals between stages XV and XX, while
this nucleus in control animals continues to increase in cell number. The left
medial nucleus of the experimental animals has undergone a significant decrease
in the cell number by stage XX compared to the number of cells in the same
nucleus of control animals.
B
Left
Control
Experimental
20000
10000
Right
20000
•Control
10000
5000
5000
Medial
;
i
I 1000
1000
500
I 500
100
100
0
1
V
X XV XX XXV
Larval stage
0
1
V
X XV XX XXV
Larval stage
Fig. 3. T o t a l n u m b e r of cells in the h a b e n u l a r nuclei of control a n d experimental
a n i m a l s : A, left medial a n d left lateral nuclei; B, right nucleus.
There is no significant difference in the number of cells populating the left
lateral habenular nucleus in the operated animals when compared to the unoperated control animals at any of the larval stages studied. At each stage
examined, however, there were significantly fewer cells in this nucleus than in
its medial companion on this side.
Fig. 3B shows the total cell population of the right nucleus, and reflects the
changes found in the length and relative volume. There is a great increase in the
number of cells contained in this nucleus between stages I and XV in both the
experimental and control animals. Between stages XV and XX in the experimental animals there is less increase in the number of cells while in the control
animals the cell numbers continue to increase. Between stages XX and XXV
there is a significant decrease in the total number of cells observed in the right
nucleus of the experimental animals when compared to the increase of the cell
number shown in the control animals. The number of cells at each larval stage
664
B. K. NORRIS AND V. B. EICHLER
examined in both control and experimental animals are clearly similar in the
right and left medial nuclei and clearly dissimilar in either of these when compared to the left lateral nucleus in the same animals.
DISCUSSION
The most complete descriptions of the neural connections of the habenular
nuclei in amphibians are given by Herrick (1948) and Frontera (1952), and both
authors indicate that the major bundles of afferent fibers enter the habenula
from areas under strong olfactory influence. Experimental proof of this association has not been shown in amphibians, although it has been demonstrated in
mammals (Rausch & Long, 1971).
To test experimentally whether the habenula complex of amphibians receives
olfactory sensory information, and to determine which of the three prominent
nuclei in this region are involved, the olfactory pits were ablated on young
embryos at an age before the olfactory nerves had contacted the brain. It is well
known that the removal of peripheral sensory end-organs results in a hypoplasia
in the corresponding neural centers (see Hughes, 1968, for a review). If any of
the habenular nuclei receive olfactory information via the nasal pits, a decrease
in size and/or cell number in the corresponding neural centers would be expected.
The experimental nature of the present study allows an extension and clarification of the functional nature of the frog habenular nuclei beyond that of
recent studies from other laboratories (Braitenberg & Kemali, 1970; Morgan
et al. 1971). It also presents an interesting discrepancy in that the two centers
reported by these authors to be homologous on the basis of morphological
appearance appear not to be the homologous pair in terms of developmental
criteria nor analogous as revealed by their functional connections. Using the
Bodian protargol method, Braitenberg & Kemali (1970) report that a fine net
of agrophilic fibers appears to course irregularly within the right and left lateral
habenular nuclei, but are absent in the left medial nucleus. We have confirmed
this observation in our laboratory (Fig. 4). However, it is clear from the results
of the present study, as will be discussed below, that it is the left medial nucleus,
and clearly not the left lateral nucleus, which develops spatially and temporally
in a manner similar to the right habenular nucleus. Moreover, the left medial
nucleus also is affected in the same way, to the same degree, and at the same
developmental age, as the right nucleus following embryonic removal of the
olfactory pits. The left lateral nucleus is not significantly affected in size nor in
cell number as a result of the experimental manipulations of this investigation.
Unfortunately, the data available at the present time do not allow a satisfactory
resolution to this discrepancy of homologies based on morphological appearance
on the one hand, and homologies based on developmental sequence and
functional connections on the other.
In our material we have found that the left lateral nucleus develops at approxi-
Fig. 4. Transverse section through the diencephalon of an adult Rana pipiens frog. Fibrous nature of right {Hr) and
left lateral (HI) nuclei may be compared with the non-fibrous appearance of the left medial (Hm) habenular nucleus.
Epiphysis (E) is indicated. Marker = 25 /*m.
•x < ^JH *
ON
I
I"
b
666
B. K. NORRIS AND V. B. EICHLER
mately larval stage V, which contrasts with the finding of Morgan et al. (1971)
who describes this cell group as a secondary development which does not form
completely as a separate nucleus until after metamorphosis. This difference
may reflect the different species of Rana frog used in the two experiments. In
Rana pipiens we find no left lateral nucleus generally observable in animals of
either the control or experimental groups before stage V; however, in two
control animals the left lateral nucleus was present to a small degree (approximately 30 jiim in length in each animal) by this early larval stage. This cell group
could thus be considered a secondary development to the medial nucleus,
which is regularly present as early as stage I, but both are present throughout
most of the larval development of this species. The left lateral nucleus was
present in all our animals at stage X, the next older stage examined.
The reduction in the length, relative volume and total cell number observed
in the right and left medial habenular nuclei of the operated animals would
indicate some important nerve connection between the peripheral sensory endorgan and the neural center. The hypoplasia noted indicates that the right and
left medial nuclei are centers receiving afferent olfactory fibers. Because the
experimental left lateral nucleus did not demonstrate any such reduction in the
three parameters measured, it appears that the left lateral nucleus is not a center
receiving afferent olfactory fibers. It is thus concluded that the right habenular
nucleus and the left medial habenular nucleus of the frog brain are homologous
and analogous structures based on their developmental pattern and functional
responses. The lateral habenular nucleus on the left side of the frog brain is
unlike these other two diencephalic nuclei in both development and functional
connections.
We have no satisfactory answer to explain why the two nuclei which are
affected by the embryonic extirpation of the olfactory pits do not show a significant response until near the end of the larval period. It has been suggested
(Norris, 1974) that the olfactory sense becomes of increased importance as the
adult stage is approached. If the semi-terrestrial frog depends more on an acute
olfactory sense than the aquatic tadpole, this would be a possible explanation,
but such data is not available at present. Risser (1914), in fact, has reported that
tadpoles of the toad react more strongly to olfactory stimulation than adults,
and Noble (1954) supports this view for amphibians in general. On the other
hand, it may be related in some way to the general maturation of the central
nervous system in middle to late prometamorphic stages in response to the
development of a functioning hypothalamo-hypophyseal system as metamorphic
climax approaches (Etkin, 1965, 1966). The latter explanation has recently been
shown (Eichler & Gray, 1975) to be valid for the prometamorphic changes in
developmental rate attending continuous illumination or darkness in larval
frogs of the same species.
The authors wish to thank Patricia Taylor for her skilled laboratory assistance.
Development of frog habenular nuclei
667
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