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The effect of endocrine gland extirpations on the size of nucleoli in rat hypothalamic neurons.

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The Effect of Endocrine Gland Extirpations on the
Size of Nucleoli in Rat Hypothalamic=Neurons ’
Department of Anatomy, Boston University School of Medicine,
Boston, Massachusetts
With the object of investigating the so-called “feed-back” mechanism
whereby hormones from the various endocrine glands presumably influence the
hypothalamus in its regulation of the hypophyseal tropic hormones, the effect of
hypophysectomy, ovariectomy, thyroidectomy, adrenalectomy and adrenal medullectomy on the nucleolar size of neurons in 16 hypothalamic nuclei was studied.
A total of forty-five female albino rats was used. The nucleoli were measured under
oil by means of a Cooke AEI image splitting eyepiece. For every rat 50 nucleoli in
each of the 16 hypothalamic nuclei was measured, a total of 36,000 measurements.
Hypophysectomy was found to affect all of the nuclei either after a short period
(5 days) or after 60 days. Ovariectomy (20 days) caused nucleolar size changes in
six nuclei, thyroidectomy (30 days) in eight, adrenalectomy (14 days) i n all of the
nuclei and adrenal medullectomy (30 days) in all of the nuclei. The results indicate
that the absence of the various hormones does affect the activity of hypothalamic
neurons as determined by nucleolar size changes. The use of the present methods
also appears to “localize” centers for specific activities in the hypothalamus in some
instances (e.g. ovariectomy) but not in others (e.g. adrenalectomy).
Numerous investigations of the relation
of the hypothalamus to the pituitary have
led to the conclusion that many of the
endocrine functions of the pituitary are
regulated in varying degrees by the hypothalamus (for reviews see Benoit and
Assenmacher, ’55; Harris, ’55). To account for the effective control of hypophyseal functions by the hypothalamus some
“feed-back” mechanism from target organ
hormones would appear to be necessary
and several recent investigations support
this hypothesis. In these studies various
technical methods have been used. TO
cite a few examples, Flerk6 and Szentkgothai (’57) grafted ovarian tissue in rats
into the hypothalamus near the paraventricular nucleus and found a significant
decrease in uterine weight as compared to
control animals. Lisk (’60) found that
estradiol implanted in the arcuate nucleus
and mammillary body resulted in prolonged diestrus in female rats. Similar
implants prevented the appearance of
pituitary castration cells (’62). Lisk and
Newlon (’63) have also shown that the
mean diameters of nucleoli of arcuate neurons are significantly smaller in the estradiol-implanted animals and a similar trend
occurred in the mammillary nucleus.
Another approach to the problem of the
“feed-back” mechanism has been that of
Sawyer and Kawakami (‘59) who studied
the effects of ovarian steroids on the EEG
of the rabbit. They considered their results to be “consistent with the concept
that hypothalamic ‘centers’ controlling sex
behavior and gonadotropic secretion may
represent important neuroendocrine receptors of hormonal influence on brain function.”
Changes in nuclear size as an indication of activity of hypothalamic neurons
following endocrine gland extirpations
and following administration of the target
hormones have been used by a group at
Pkcs University Medical School in Hungary to study the relations between the
endocrine gland and. the hypothalamus.
Their results have been summarized in a
monograph ( SzentAgothai, Flerk6, Mess,
and Halhsz, ’62) and some of the details
of their work will be considered in the
discussion to follow. They found, in general, that the endocrine glands profoundly
influenced the activity of the hypothalamic neurons. These authors, however,
do not attempt to assign any specific functional state (hyper or hypoactivity) to
nuclear size changes but consider these
changes as merely an indication of some
kind of neuronal activity.
XSupported by a grant from the National Science
cleoli in each of the 16 hypothalamic
nuclei. This median figure for each nucleus was then set on the eyepiece dial
and in every microscopic field examined
the nucleoli were seen to be either smaller
or larger than, or equal to, the median
size. To illustrate, the median, expressed
in arbitrary units of the eyepiece, for the
supraoptic nucleus was 4.262 and this
dial setting was used thereafter for supraoptic nuclei measurement. Fifty supraoptic nucleoli then were counted for each
rat and each nucleolus was placed in one
of the three size categories. Thus in measForty-five female albino rats (approxi- uring the nucleoli in the supraoptic numately 250 gm each) from the Charles cleus of ten normal rats 41 nucleoli were
River Breeding Laboratory were used in found to be larger than the median, 207
these experiments. Ten were used as con- equal to the median and 252 smaller than
trols, ten were hypophysectomized and the median. The nucleoli of ten animals
killed after five days, five were hypophys- hypophysectomized for five days were comectomized and killed after 60 days, five posed of 34 larger, 169 equal to, and 297
were thyroidectomized and killed after 30 smaller than the median. Whether this
days, five were ovariectomized and killed “shift” to the smaller size after hypophysafter 20 days, five were adrenalectomized ectomy was statistically significant or not
and killed after 14 days, five were adrenal was determined by the chi-square test.
medullectomized and killed after 30 days. This procedure was then followed for all
The animals were killed by decapitation; of the measurements. Where more than
the hypothalamus was removed and fixed one operator was making measurements
in Hollande’s solution, sectioned in paraf- each operator measured an equal number
fin at 10 ~1 and stained with cresyl violet. of controls and experimental animals.
All extirpations were checked for comRESULTS AND DISCUSSION
pleteness at autopsy and in the adrenal
Table 1 summarizes the results of the
medullectomized rats the remaining adrenal cortex was sectioned and examined chi-square test for significance with P
microscopically for the presence of medul- values of less than .05 being considered
lary tissue. Only those animals were used significant. The direction of the “shift”
in nucleolar size is indicated also; a (+)
in which the extirpation was complete.
The nucleoli were measured under oil indicating an increase in the number of
by means of a Cooke AEI image splitting nucleoli larger than the median size, a
eyepiece (Dyson, ’60). For every rat 50 (-) indicating an increase in the number
nucleoli in each of 16 hypothalamic nu- of nucleoli smaller than the median size.
clei (listed in table 1, terminology of Rioch In the following discussion of the results
et al., ’40) was measured, a total of 36,000 the expression “nucleoli were enlarged sigmeasurements. The suprachiasmatic nu- nificantly” or ‘%ad larger nucleoli” refers
cleus was omitted (Ifft, op. cit.). Nucleoli actually to an increase in the number of
of so-called “dark” neurons when seen these larger nucleoli. Similarly an increase
were not measured although Cammer- in the number of smaller nucleoli is remeyer (’62) considers that the nucleolus ferred to as a decrease in nucleolar size.
remains unchanged in these otherwise
It will be seen from table 1 that hypophshrunken cells.
ysectomy for a short period of time (five
A comparative measurement technique days) resulted in nucleolar size changes in
was used. In this method a median size 13 nuclei. Of these PV, SO, VM, MM, and
for the nucleoli was established by meas- ML had a shift toward smaller nucleoli
uring and finding the median for 100 nu- while the other nuclei changed in the opIn the present work the effect of hypophysectomy, ovariectomy, thyroidectomy, adrenalectomy and adrenal medullectomy on the nucleolar size of neurons
in 16 hypothalamic nuclei was studied.
Nucleolar size as a criterion of nerve cell
activity was used in a previous investigation and its validity considered there
(Ifft, ’62) in comparison with the criterion
of nuclear size. An increase in nucleolar
size is considered to be an indication of
increased activity of the neuron.
posite direction. Since PV and SO have
direct nerve connections with the neurohypophysis, injury to their axons might
be expected to result in a temporary decrease in function. However, after 60
days of hypophysectomy, PV and SO had
changed to enlarged nucleoli but the total
number of neurons could be seen by inspection to be greatly decreased (cf. Rasmussen, ’40). Cell counts were not made
on the other nuclei with decreased nucleolar size : the possibility exists that they
were affected by axonal damage. In animals hypophysectomized for 60 days, each
of these nuclei had either returned to normal nucleolar size or the nucleoli were
enlarged significantly. No nuclei were unaffected by hypophysectomy although
some showed changes after hypophysectomy of five days duration only, others
after 60 days only.
Ovariectomy affected six nuclei, the
smallest number influenced by any of the
extirpations. This suggests the possibility
of “localizing” the gonadotropic centers of
the hypothalamus to some extent. It would
appear that SO and PV were involved (cf.
Matsui and Engelhardt, ’60) but these
nuclei were affected by nearly all of the
other extirpations. Considering the results
of previous studies (e.g. Ifft, op. cit.;
Olivecrona, ’57) it is questionable whether
these nuclei are concerned with gonadotropic activities yet it appears that they
respond to a decrease in estrogen. The
arcuate nucleus had smaller nucleoli following ovariectomy. This decrease is unexpected since arcuatus has been considered a gonadotropic center by several
investigators (see introduction). However
Szent5gothai et al. (op. cit.) reported no
change in arcuatus following ovariectomy
but found estrogen increased the size
of the nuclei of arcuatus’ neurons. The
decrease in nucleolar size reported here is
contrary to expectations since estrogen
has been found to decrease nucleolar size
(Lisk, op. cit.). A possible explanation is
that the feed-back mechanism may be
complex with both gonadotropins and estrogens acting on the hypothalamus. Studies are in progress to test this hypothesis.
Diagrams of such possible complex feedback mechanisms recently have been published (Scharrer and Scharrer, ’63).
Ovariectomy also affected the mammillary region except for ML. This is in
agreement with Szentdgothai et al. (op.
cit.) and also with Lisk (op. cit.). A
nucleolar size decrease occurred in MM as
was noted for arcuatus. The remainder of
the nuclei were not changed by ovariectomy. It is surprising that AA remained
normal since lesions in this region produce constant estrus (Hillarp, ’49).
SzentAgothai et al., found a reduction of
nuclear size in this region following ovariectomy but also after estrogen administration. With such conflicting evidence it
does not seem possible to assess the exact
role of AA in the estrous cycle.
Thyroidectomy resulted in nucleolar enlargement in seven nuclei, and a nucleolar
decrease in size in one nucleus, MM.
Szentdgothai et al. found nuclear enlargement in PV, a decrease in PMA, and no
change in the other nuclei reported here.
These authors also include the habenular
nuclei in their study and found a significant decrease in nuclear size in this area
which they concluded was an important
region for thyroid control. Recently Matsuda (’63) found that bilateral ablation of
the habenular nucleus had no effect on the
thyroid of the rat.
It is of interest that these seven nuclei
respond to thyroxin deficiency by an increase in nucleolar size. Apparently the
expected generalized decrease in metabolic
activity accompanying thyroidectomy does
not affect most hypothalamic neurons (MM
had a nucleolar size decrease) and therefore these nuclei showing nucleolar enlargement may be concerned with the control of thyroid-stimulating hormone release. However, the nuclei listed here correspond only in part to the “thyreotropic
region” described by others and localized
by means of lesions, or electrical stimulation, or injections of thyroxine into the
hypothalamus (e.g. Yamada and Greer,
’59; Okinaka et al., ’60; Averill et al., ’61).
PV is included in the area by most investigators as it is here. The more posterior nuclei which were found responsive in
the present experiments are not usually
included in the “thyreotropic region” by
others. Again it must be concluded that
much remains unknown about the relation of the hypothalamic nuclei to thyreo-
Results of chi-square test for significance between the experimental groups and
the normal for each hypothalamic nucleus
(5 days)
Preoptic area (POA)
preopticus (PP)
Anterior area (AA)
Lateral area (LA)
Supraopticus diffusus (SD)
- <0.005
Paraventricularis (PV)
- <0.02
Supraopticus (SO)
Arcuatus (A)
- <0.02
Ventromedialis (VM)
Dorsomedialis (DM)
Dorsal area (DA)
Posterior area (PA)
Premammillary area (PMA)
Supramammillary area (SMA)
- <O.Ol
Mamillaris medialis (MM)
- <0.005
Mamillaris lateralis (ML)
(60 days)
(30 days)
(14 days)
( 30 days )
+ <0.005
+ <0.005
+ <0.025
+ <O. 005
+ <0.005 + <0.005
+ <a005
+ <0.005
+ <0.005 + <0.005
+ <0.005
- + <0.005
+ <0.005
+ <0.005 + <0.005
+ <0.005
+ <0.005
- + <0.005
+ <0.005
+ <0.005
- + <0.005
- + <0.005
+ <0.005 + <0.005
+ <0.005 + <0.005
+ <0.005 + <0.005
- <0.005 + <0.005
- + <O.O05
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <O.OOS
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <0.005
+ <0.005
- <0.05
+ + <o.oos
+ <0.005 + <0.05
+ <0.005
+ <0.005
- <0.05
(+) and (-) signs indicate direction of “shift” (see text for explanation).
P values in chi-square test. P is considered significant when < 0.05.
3 P values > 0.05 not given.
tropic activity of the pituitary. Some of
the questions remaining to be answered
have been discused recently by Bogdanove
Adrenalectomy resulted in significant
changes in all of the nuclei: adrenal medullectomized animals also had significant
size increases of the nucleoli of all the
nuclei so that it is not possible from this
experiment to distinguish between cortical
and medullary effects. SzentLgothai et al.
(op. cit.) found widespread hypothalamic
changes following adrenalectomy. They
considered these changes to be the result
of the removal of the cortex although they
did not perform adrenal medullectomies
in their experiments. Their conclusions
was based on their observations that the
same hypothalamic changes could be induced by interruption of the nerves from
the adrenal glands as could be obtained by
adrenalectomy or atrophy following cortisone administration. The adrenal medulla
was considered not to have a role since
cortisone treatment does not produce
significant changes there.” The interruption of nerves from the adrenal cortex was
thought to be the important factor. They
further report and consider crucial evidence that unilateral adrenalectomy causes
cell nuclei enlargement in VM on the con‘ I . .
tralateral side of the extirpated adrenal
while on the same side as the remaining
hypertrophied adrenal the VM nuclei are
smaller. Further discussion of their theory
may be found in the original paper (HalLsz
and SzentLgothai, ’59). Since in the medullectomized animals in the present experiment the cortical innervation was not
examined, further comment on the above
hypothesis will be deferred until additional
evidence is obtained.
The results of the experiments reported
here show that the absence of the various
hormones does affect the activity of hypothalamic neurons. It would appear also
that some degree of localization in the hypothalamus is possible by use of the present methods as suggested by the few nuclei found to be affected by ovariectomy.
Adrenalectomy, on the other hand, resulted
in changes in all of the hypothalamic nuclei. In this case further refinements of
experimental procedures will be required
if ‘localization” is to be obtained. However, it may very well be that the whole
hypothalamus is involved in regulation of
many of the endocrine functions and that
the association of hypothalamic control
with specific nuclei is not always possible.
This viewpoint also has been expressed by
SzentAgothai et al.
The able technical assistance of Lynda
Simoneit and Raymond Pong is gratefully
acknowledged as is the helpful advice of
Dr. E. T. Angelakos on statistical analysis.
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effect, endocrine, extirpation, hypothalamus, gland, size, rat, neurons, nucleoli
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