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The effect of temperature differentials upon the differentiation of Mauthner's cell in Ambystoma.

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The Effect of Temperature Differentials upon the
Differentiation of Mauthner's Cell
in Ambystoma '
JEAN PIAT'T
Department of Anatomy, School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
ABSTRACT
Eggs of Ambystoma maculatum were separated into three groups
and maintained at 5", 8" and 14"C,respectively, until stage 27. At this stage the
otic vesicle was extirpated from the right side, thus eliminating the VIIIth cranial ganglion and root. The number of cases in which Mauthner's cell (MC)
failed to differentiate in the absence of VIIIth roots was compared among the
three temperature groups.
In a total of 713 animals, MC was absent on the side of operation in 200 cases
(28.05% ). Comparison of the three temperature groups with respect to the absence of MC is as follows: 14", 39 of 212 cases (18%); 8", 83 of 266 cases
(31%); 5", 78 of 235 cases (33% ). Statistical analysis of these data demonstrates a significant difference between either one of the colder groups and the
14" group with regard to suppression of MC. When the two colder groups are
tallied as a single sample, the difference is even more significant. There is no
significant difference between the 5" and 8" groups. These results indicate that
the differentiation of MC is more dependent upon the ingrowth of VIIIth root
fibers when morphological development has been delayed by prolonged hypothermia.
Recent experiments (Piatt, '69) provide
ample evidence that the differentiation of
Mauthner's cell (MC) in the medulla oblongata of the salamander (Ambystoma
maculatum) is dependent to some extent
upon the presence of the VIIIth cranial
nerve root (vestibular). Unilateral removal
of the primordium of the vestibular ganglion resulted in absence of MC on that
side in 27.8% of all cases. In addition, it
was shown that the VIIth lateral-line
root fibers also exert a developmental
stimulus to the differentiation of MC. Earlier studies (Piatt, '43, '47, '50) also lend
support to these results.
This same series of experiments yielded
further data that suggested a causal relationship between greater chronological age
(slower rate of development) and greater
influence of the VIIIth root upon the differentiation of MC. In certain series, for
instance, when the VIIIth root was absent,
following unilateral ablation of the otic
vesicle at stage 27, MC failed to differentiate in 35.8% of the cases in those aniANAT. REC.,169: 41-44.
mals which had been maintained at a temperature of 8°C prior to operation, as opposed to 16.6% failure in animals raised at
14°C prior to operation. Statistical analysis
of these data indicated that the difference
between the two groups might be significant (p slightly > 0.01). On the other hand,
there was no significant difference between
other similarly raised groups of animals
and there was no consistent difference between groups raised at 5" and 14°C. Furthermore, in assembling and comparing
the data among the different temperature
groups no distinction was made between
those cases in which MC was absent when
only the VIIIth roots were missing and
those cases in which both the VIIIth and
VIIth lateral-line roots were missing. It was
concluded that the entire series of experiments represented too small a sample from
which to assess adequately the effects of
temperature differentials (differences in
Received April 27, '70. Accepted July 15, '70.
'This research was supported by grant 1ROl-NB
08112-01 from the National Institutes of Health.
41
42
JEAN PIATT
rate of development) upon the suppression
of MC in the absence of the VIIIth root.
The present study is a necessary extension
of the experiments outlined above. It
furnishes reliable evidence that differences
in rate of development affect the differentiation of MC with respect to its dependency upon the presence of VIIIth root
fibers.
PROCEDURE
Ambystoma maculatum was the species
used in these experiments. Temperature
control of developing embryos prior to OPeration was carried out as follows. Each
clutch of eggs was divided into three equal
groups as soon as they reached the laboratory. Each group was maintained continuously at temperatures of 5", 8" and
14"C, respectively, until stage 27 was
reached. The stages at which temperature
differentials were initiated were 1, 3, 4, 5,
7 and 9. The various series within each
temperature group were identified by
clutch as well as temperature so that possible differences among the clutches could
be noted. Following removal of the otic
vesicle, all groups were maintained at an
approximate temperature of 18°C.
The otic vesicle (primordium of ear and
VIIIth ganglion) was extirpated from the
right side of the embryos at stage 27 in
all three temperature groups. Extreme care
was exercised to remove just that ectoderm
immediately adherent to the otic vesicle in
order to eliminate only vestibular root fibers. Despite these precautions, VIIth
lateral-line roots failed to develop in a large
number of cases although some ganglion
cells were always present. All animals were
fixed at stage 46. The entire head was
serially sectioned and impregnated with
silver on the slide. A total of 1019 animals
was examined for: ( 1 ) presence or absence of MC, (2) presence or absence of
vestibular and lateral-line roots, ( 3 ) and,
especially, the respective absence of MC
among the three temperature groum employed. Both the VIIIth and VIIth lateralline roots were absent in 288 cases and
have been excluded from this report.
Eighteen animals in which a few VIIIth
root fibers developed are also excluded. The
data of this paper, therefore, are based
upon a total of 713 animals.
RESULTS
Mauthner's cell failed to differentiate on
the side of operation in 200 animals or
28.05% of all cases. Table 1 summarizes
the essential data upon which an analysis
of the differences among the three temperature groups is based. In 235 animals
maintained at 5°C prior to removal of the
otic vesicle, MC failed to differentiate in
78 animals or 33.19% of the cases. The
average time spent at 5" for all the series
within this group was 849 hours, with extremes of 714 and 1051 hours depending
upon the stage of development at which
temperature differentials were initiated. In
266 animals maintained at 8", MC was absent in 83 animals or 31.2% of the cases.
The average time spent at 8" was 463
hours, with extremes of 378 and 598 hours.
In 212 animals maintained at 14"C, MC
was absent in 39 animals or 18.39% of
the cases. The average time spent at 14"
was 183 hours, with extremes of 137 and
21 1 hours.
Statistical analysis of these data demonstrates a significant difference between
either one of the colder groups and the
14" group with regard to suppression of
MC, following removal of the otic vesicle.
When the 5" and 8" groups are tallied as a
single sample, the difference between the
colder and warmer groups is even more significant. There is no significant difference
between the 5" and 8" groups. When the
5" group is compared with the 14" group
with respect to the absence of MC, p is less
than 0.001. When the 8" group is compared with the 14" group, p is much less
than 0.01. When the two colder groups are
considered as a single sample, p is decidedly less than 0.001. When the 5" and
8" groups are compared with each other, p
is approximately 0.7. The Chi-square values for the determination of these probabilities are given in table 1. Prolonged
hypothermia, resulting in a much slower
rate of morphological development, has in
some way affected the differentiation of
MC so that the cell is more dependent upon
the ingrowth of vestibular root fibers than
when the rate of development is faster.
There were no consistent or significant
differences among the different clutches of
eggs with respect to the relative number of
cases in which MC was absent within any
43
DIFFERENTIATION OF MAUTHNER'S CELL
TABLE 1
Summary of the results of the differentiationof Mauthner's cell among the
three temperature groups in the absence of VIIIth root fibers
MC absent
Temperature
groups
~
Number of
animals
Average time in
hours before
operation
Cases
235
266
212
849
463
183
78 (33.19)
83 (31.2)
39 (18.39)
~~~
~
~
Chi-squarevalues for
differences between
temperature groups
%
5O
8"
14"
713
one temperature group. It would appear
that, at least up to stage nine in development, hypothermal effects are about the
same for all stages through early cleavage
to blastula with regard to relative suppression of MC in the absence of the VIIIth
root.
DISCUSSION
The nature of the influence exerted by
vestibular root fibers on the differentiation
of MC is quite obscure. To the best of the
author's knowledge, there are no electrophysiological or biochemical studies that
bear directly on this subject. With the exception of two series, one in the 5" group
and one in the 8" group, MC still differentiated in the majority of the cases within
each temperature group, despite the absence of the VIIIth root. Other factors, obviously, are at work. The reader is referred
to a former study (Piatt, '69) for a brief
discussion of this problem.
The difference between the colder groups
and the 14" group with regard to the differentiation of MC is the chief concern of this
particular study. The data have been assembled from a large number of animals
and their statistical validity has been demonstrated. This being the case, an intriguing problem presents itself; namely what
basic cytological processes have been altered within the precursor of MC and in
what manner? The situation is undoubtedly
complex and at its core is the basic problem as to whether the biochemical processes of development can be altered in
their sequential elaboration by decreasing
or increasing rates of development in such
a manner that the visible, morphological
events of development no longer constitute
a reliable index of the actual range of de-
200 (28.05)
5" cf. 8" = x2 0.144
8"cf. 14"= xa 9.51
5"cf. 14"= xe 11.87
(5'+8')
cf. 14"= x 2 13.2
velopmental potentialities. In other words,
can the biochemical and morphological
events of development be dissociated in
time? Previous experiments by the author
and others indicate that they can and the
reader is referred to an earlier paper (Piatt,
'68) for a brief discussion of this problem
and a review of some of the pertinent literature. The results of the present study
point in the same direction; namely, under
the attendant experimental conditions, a
stage 27 Ambystoma embryo raised at 5" or
8" does not have the same potentialities for
development with regard to the differentiation of MC as it does when raised at 14°C.
One can only surmise that a slower rate
of development, caused by hypothermia,
has in some way affected the initial processes of differentiation in the precursor
of MC to such an extent that the cell i s
more dependent upon the impetus afforded
it by ingrowing vestibular root fibers. Unfortunately, the character of the present
data affords no clue as to the actual nature
of these early alterations.
LITERATURE CITED
Piatt, J. 1943 The course and decussation of
ectopic Mauthner's fibers in Amblystoma punctatum. J. Comp. Neur., 79: 165-183.
1947 A study of the factors controlling
the differentiation of Mauthner's cell in Amblystoma. J. Comp. Neur., 86: 199-235.
1950 Further studies on the differentiation and growth of Mauthner's cell in Amblystoma. J. EXP. ZOO^., 113: 379-395.
1968 Dissociability of developmental
processes in the morphogenesis of the ear in
Ambystma, as revealed by x-ray damage. Anat.
Rec., 160: 143-151.
1969 The influence of VIIth and VIIIth
cranial nerve roots upon the differentiation of
Mauthner's cell in Ambystoma. Devel. Biol.,
19: 608-616.
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