Attempts were made to identify hypothalamic neurosecretory cells which participate in the anterior pituitary control with a special reference to the effects of ovarian hormones. Wave forms of the antidromically activated action potentials in the hypothalamus were investigated in the female rats of Wistar strain. The animals of different endocrinological environments, that is, proestrus and diestrus-I of 4-day cyclers; ovariectomized, ovariectomized and estrogen primed, ovariectomized and progesterone primed, were used. The operational procedures were performed under light urethane anesthesia. Different two types of craniotomy were adopted for the preparation. For recordings from the medial basal hypothalamus, the ventral surface of the brain was exposed by parapharyngeal approach, and a bipolar silver ball electrode for the application of electrical stimuli was placed on the surface of the median eminence (ME). Another method was used for recordings of the medial preoptic area (MPO) and the periventricular area of the MPO and the anterior hypothalamus (PVA). In the latter, the parietal surface of the brain was exposed and a side-by-side bipolar electrode for the stimulation was inserted to the arcuate (ARC) -ME region.
All the recordings were performed by using glass pipettes which were filled with 0.5 M sodium acetate solution. The solution was added with pontamine sky blue 6B to allow marking recording sites. Stimulatory pulses of 5 msec duration of variable intensity were generated by an electronic stimulator and applied by the above mentioned bipolar electrodes. The antidromic responses and the intensity of the stimuli were observed and photographed on a synchroscope.
The following results were obtained :
(1) Antidromic responses were recorded from the ARC to the ME stimulation (n=97). The ME-surface stimulation was also effective in inducing antidromic responses in the PVA and 12 responses were observed by this method. The ARC-ME stimulation resulted in 74 antidromic responses in the MPO, and 106 responses in the PVA. These responses showed constant latency and followed to the high frequency stimulation and indicated that these cells send their axons directly to the stimulated sites.
(2) The conduction velocity of the axon was calculated from the latency and the distance between the recording and stimulating sites. The calculated velocities were : about 0.2 m/ sec for the ARC cells; 0.4 m/sec for the MPO cells, and 0.3 m/sec for the PVA cells.
(3) Most of the responses in the ARC and PVA showed positive-negative biphasic wave form, with a notch in the positive-going phase of each spike, whereas no notch was seen in the all of responses obtained from the MPO.
(4) In the ARC and the PVA high frequency repetition of the stimulation induced fractionation of small A-spike and large B-spike at the notch. A-spike component followed exactly to the stimulation of 100 Hz, but a delay or inhibition of B-spike component was occasionally seen to the repetition higher than 10 Hz.
(5) Fractionation of A-, and B-components were seen to be affected by hormonal environments of the animal. In the proestrus, B-spike inhibition was not seen in 5 Hz repetition of the stimulatory pulses, whereas in the diestrus-I, inhibition of B-spike was seen in 2 cases among 10 trials with 0.5 Hz repetition. In the ovariectomized rats, estrogen priming increased responsiveness of B-spike formation to high frequency stimuli. On the other hand, suppression of B-spike formation was seen following progesterone treatment.
(6) In order to investigate the possible mechanisms of the fractionation of A- and B-components, the effects of train stimulation of 10-100 Hz in the ARC-ME was investigated. Spontaneous activity of the PVA units were seen to be suppressed by the stimulation, not only the B-spike but also the A-spike. Suppression of B-spike amplitude was observed by applying hippocampal stimulation prior to the ME,

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Attempts were made to investigate the chemical nature of hypothalamic afferents from the extrahypothalamic structures which participate in the control of hypothalamic neurosecretory cells. Experimental procedures were discribed in the preceding report, except that of the electrical stimulation and the microiontophoresis. Some part of data obtained in this study were referred in the preceding report for improvement of statistic reliability. For electrical stimulation, side-by-side bipolar electrodes were placed in the medial preoptic area (MPO), medial amygdala (mAMYG) and dorsal hippocampus (dHPC) from the parietal surface of the brain and fastened to the cranium by dental resin. Microiontophoresis of norepinephrine (NE), dopamine (DA) and acetylcholine (Ach) was carried out by using five-barrelled glass pipette on the antidromically activated units in the medial basal hypothalamus by the median eminence stimulation. Extracellular potentials were recorded by the central barrel of each electrode. One of the outer barrels was filled with physiological saline and used for control purpose.
(1) Electrical stimulation of the MPO with train pulses of 0.2 msec duration and 300 μA intensity in 100 Hz for 5 sec, induced facilitation and inhibition in 26.4% and 9.4% of 106 tests, respectively. By the mAMYG stimulation with 500 μA, facilitation was seen in 18.8% and inhibition in 10.4%, of 96 tests. The HPC stimulation induced facilitation in 10.0% and inhibition in 23.3% of 90 tests.
(2) Facilitatory effect of the MPO was most frequently seen in the ovariectomized and estrogen primed rats (58.8% of the tests). In contrast, the inhibitory effect of the dHPC was striking in the diestrus-I (40.0%).
(3) Microiontophoresis of NE induced facilitation in 60.0% and inhibition in 10.6 % of 66 identified cells. DA induced facilitation in 45%, and inhibition in 11.7 % of 60 tests. Ach induced facilitation in 41.4%, and inhibition in 13.8% of 58 tests.
(4) By NE infusion, facilitation was most commonly seen in the proestrus (70.6 %). Inhibition was most readily elicited by Ach infusion on the diestrus-I (30 %). Facilitative effect of DA was eminent in the diestrus-I (81.8%).
(5) Successive administration of NE and DA on identical units revealed that in 56.0 %, the units were responsive to only one agent and the other was effectless. 25.5% of the units responded to both of NE and DA.
(6) The coinceidence of effects, either facilitation or inhibition, between that of electrical stimulation and of the microiontophoresis, was calculated on 46 units. The facilitatory effect of the MPO was most commonly mimicked by NE infusion (81.8 %), and the inhibitory effect of the dHPC by Ach infusion (46.7%). NE also mimicked facilitatory effect of mAMYG stimulation (73.3%).
(7) Intraventricular infusion of 20 μg of NE induced significant increase of serum LH (p<0.05 to saline) when measured by radioimmunoassay, whereas DA resulted in a decrease of LH (p<0.01).

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In 1936, Ehrhadt reported a prolactin-like substance which has been identified as a second protein hormone exists in extracted substance from the placenta. To date various reports have been published, but there are still unsolved problems concerning the specifity of the substance, and its clinical significance is unclear. The purpose of this project was to purify and investigate the biochemical, immunological properties of HPL, and try to localize HPL secretion immunohistologically.
The biological properties examined by intramuscular and intradermal pigeon crop sac reactions, both showed positive results. Biochemical properties were tested by 7.5 % polyacrylamide disc electrophoresis and Free Fatty Acid mobilization. By these methods, the former showed a single band by electrophoresis with purified HPL 200 μg, and the latter showed a strong positive reaction both in vivo and in vitro. Immunological propertiese were examined by immunoelectrophoresis and the Ouchterlony method. In immunoelectrophoresis, the reaction of purified HPL 80 μg and anti-HPL serum resulted in a single strong zone. In contrast, anti-serum and HGH resulted in a weak zone of reaction. We confirmed that HGH cross reacts with our anti-HGH serum to HPL. The cross reaction of HGH with anti-HPL serum was reported by Josimovich et al, and other workers. In the Ouchterlony method, no precipitation line was detected between anti-HPL serum and 50-300 iu of HCG (PREGNYL). The results of investigating the localization of secrtions of HPL immunohistologically, show that the specific fluorescence was limited to the strong reacting syncytial layer in the both the early chorionic villi of normal pregnancy and in the late stages of pregnancy. And the results of examining the localization with enzyme reactions with peroxdase from horseradish Type II, the reaction of benzidine brown was limited to the syncytial trophoblastic layer as in the case of FITC. These results suggests that the localization of the secretion of HPL exists in the syncytial layer of the chorionic villi.

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Hypothalamic-pituitary-adrenocortical function was investigated in 14 patients with anorexia nervosa. Impaired suppression of plasma cortisol by dexamethasone was revealed. In 14 patients with anorexia nervosa, circadian rhythm of plasma cortisol, insulin tolerance test, rapid ACTH test and overnight dexamethasone suppression test were examined.
Levels of plasma cortisol were higher than those in control subjects through out the day, and normal circadian rhythm of plasma cortisol was not observed. Basal levels of plasma ACTH were within normal range. The response of plasma cortisol to insulin-induced hypoglycemia was lower than that in control subjects, while the response of plasma cortisol in rapid ACTH test was normal. In overnight suppression tests, in which one mg dexamethasone was administered orally, 11 of 14 patients showed no suppression of plasma cortisol and 3 other patients showed incomplete suppression.
Elevated levels of plasma cortisol and the absence of normal circadian rhythm in patients with anorexia nervosa and malnutrition have already been reported by other investigators, and these abnormalities were ascribed to the delayed half life of plasma cortisol due to impaired cortisol metabolism.
However, according to our investigation, it is difficult to explain the failure of dexamethasone to suppress cortisol only by the delayed half life of plasma cortisol, and it is supposed that some kind of abnormal hypothalamic control is also involved.

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The radioimmunoassay for LH-RH would aid greatly in the assessment of hypothalamic function. The anti-LH-RH was prepared by immunizing rabbits with LH-RH conjugate of BSA. ¹²⁵I-LH-RH was prepared by the lactoperoxidase method and Sephadex G-10 column chromatography. The double antibody RIA technique was employed. On the assay of biological materials, LH-RH was extracted by methanol because LH-RH was rapidly destroyed in the serum, and this breakdown could not be prevented by benzamidine or 2, 3-dimercaptopropranol. Sensitivity of this RIA system ranged from 10 to 10⁴ pg/ml, and the coefficient of variations of intra- and interassay were 12.9% and 9.0% respectively.
The serum LH-RH levels of men in a normal gonad state were below 10 pg/ml, and those of women in a normal gonad state in the early follicular or luteal phase were below 50 pg/ml. Whereas those of postmenopausal or castrated women were increased, and were highest in women wit h several gonadal disturbed states.
The disappearance curve of LH-RH in women was characterized by tw o exponentials, t (1/2) of the initial component was 4.9 min, and that of the second component 24.6 min.
Urinary excretion of exogenously administered LH-RH was also studied.

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Effects of short and long term administrations of an oral contraceptive preparation on the pituitary responses to the synthetic LH-RH were compared in the present experiment.
Five normal cycling women were used as controls in the late proliferative phase. Five healthy volunteers taking an oral contraceptive agent (1.0 mg norethindrone with 0.05 mg mestranol) for less than 10 cycles were served as short term group and other 4 women taking the same preparation for more than 40 cycles were studied as long term group. Volunteers in each group were kept fasting overnight and 5 ml of blood was drawn at 9 to 10 am on the day of experiment. Ten minutes after drawing blood, 200 μg synthetic LH-RH was injected subcutaneously in the late proliferative phase in the normal cycling women. According to the same procedure, women taking pills were administered LH-RH on the 7th to 12th day after the beginning of taking the first tablet in each cycle. Three ml of blood was taken at 15, 30, 60,120 minutes and 24 hours, and serum LH and FSH were determined by the double antibody radioimmunoassay. The 2nd IRP-HMG was used as the standard materials and expressed as mIU/ml of serum.
Mean baseline serum LH and FSH concentrations were not suppressed in a short term administration of norethindrone-mestranol combination. Fifteen minutes after the subcutaneous injection of LH-RH, mean LH level was significantly elevated, and thereafter the level was not significantly changed as compared with that seen in the late proliferative phase of the cycle. Concerning the FSH response to LH-RH, a short term administration did not induce a significant rise. On the other hand, mean baseline serum LH and FSH concentrations were inhibited in long term use of the agent, and mean LH and FSH levels were always markedly suppressed after the administration of LH-RH. In a short term treatment the mean net increase of LH above the baseline was significantly greater than that of the control at 15 minutes after the injection of LH-RH, but at all other points the mean net increases of LH and FSH following LH-RH were not markedly changed. Moreover, the maximum FSH increase occurred earier than that seen in the late proliferative phase of the cycle. In a long term treatment, the net increases of LH and FSH were significantly lowered as compared with the control.
In terms of percent changes from baseline level, the initial LH release within the first 30 minutes after LH-RH was distinguishable from that in the control group, but thereafter the percent chages in both treated groups and in women in the late proliferative phase were not distinguishable. However, the percent increase over the baseline level of serum FSH after the injection of LH-RH was not significant at all points.
From these results, it was suggested that norethindrone-mestranol combination exerted a direct action on pituitary gonadotropin response to LH-RH. An enhansement of pituitary response to LH-RH may be due to an effect of estrogen or estrogenic activity of norethindrone-mestranol combination. In women treated in a long term, the de-creased response of both LH and FSH was most likely a reflection of an decreased pituitary gonadotropin storage, al least redily releasable component. However, the increment of the percent changes after LH-RH administration was seemed to suggest that a concomitant increase in the sensitivity of the gonadotropin producing cell (especially LH gonadotroph) to LH-RH also existed.

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In order to investigate the effect of calcitonin (CT) on calcium and phosphorus metabolism in primary hyperparathyroidism (PHP), porcine calcitonin (80 MRC units) was injected intramuscularly at 9 : 00 a.m. and 5 : 00 p.m. for 10-14 days in 7 patients with parathyroid adenoma. Fasting blood specimens were drawn at 8 : 00 a.m. every other day and 24 hour urine samples were collected through out control and test days. To examine the acute effect of CT, blood and urine were checked several times until 8 hours after the first injection.
A fall in the fasting serum calcium level observed in 5 patients during the repeated administrations of CT, as well as that observed in 6 patients within 6 hours after the first injection, showed a significant correlation with the initial serum calcium level. Serum phosphorus concentration decreased in all patients 6 hours after the first injection, while fasting levels seemed to remain unchanged.
During the repeated administrations, urinary excretion of calcium and phosphrus decreased correspondingly with the fall in serum calcium levels, although no definite tendancy was observed within 8 hours after the first injection.
Fasting serum PTH levels during the repeated administrations were measured in 2 patients. In a patient whose serum calcium returned to the initial level on the 7th day of administration, a gradual rise of PTH was observed, while in another patient whose serum calcium was kept lower than the initial level, PTH remained almost unchanged.
These results indicate that, under sucha condition where there is marked increase of bone resorption as PHP, repeated administrations of CT bring about not only a hypo-calcemic effect but also the reduction of calcium and phosphorus excretion through a decreased filtered load. In addition, it was suggested that, in some cases of PHP, the hypocalcemic effect of CT may be abolished by an increase of PTH secretion from the parathyroid glands during long-term administration.

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