The effect of endocrine gland extirpations on the size of nucleoli in rat hypothalamic neurons.код для вставкиСкачать
The Effect of Endocrine Gland Extirpations on the Size of Nucleoli in Rat Hypothalamic=Neurons ’ JOHN D. IFFT Department of Anatomy, Boston University School of Medicine, Boston, Massachusetts ABSTRACT 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 Foundation. 599 600 JOHN D. IFFT 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 MATERIALS AND METHODS 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. ENDOCRINE EFFECTS ON HYPOTHALAMUS 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). 601 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- 602 JOHN D. IFFT TABLE 1 Results of chi-square test for significance between the experimental groups and the normal for each hypothalamic nucleus Hypophysectomized (5 days) +1<0.0052 Preoptic area (POA) Periventricularis <O.Ol preopticus (PP) <0.005 Anterior area (AA) <0.005 Lateral area (LA) <0.005 Supraopticus diffusus (SD) - <0.005 Paraventricularis (PV) - <0.02 Supraopticus (SO) <0.02 Arcuatus (A) - <0.02 Ventromedialis (VM) -3 Dorsomedialis (DM) <0.02 Dorsal area (DA) Posterior area (PA) Premammillary area (PMA) <0.005 Supramammillary area (SMA) - <O.Ol Mamillaris medialis (MM) - <0.005 Mamillaris lateralis (ML) + + + + + + + OvariHYPOP~Yectomized sectomized (20 days) (60 days) Thyroidectomized (30 days) Adrenalectomized (14 days) Adrenal medullectomized ( 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 + <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 <0,005 - <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. 1 2 tropic activity of the pituitary. Some of the questions remaining to be answered have been discused recently by Bogdanove (’62). 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. ENDOCRINE EFFECTS ON HYPOTHALAMUS ACKNOWLEDGMENT 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. LITERATURE CITED Averill, R. L., H. D. Purves and N. E. 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