Developmental changes in hypothalamic melatonin levels of male rats.

Hypothalamic melatonin levels of Long-Evans male rats were studied at three ages (25, 55-60 and 90 days), at four times of the day in the autumn (6:00, 12:00, 18:00 and 24:00), and at two times (12:00 and 24:00) in the spring using radiommunoassay. Melatonin levels increased markedly at noon at 55-60 days of age, compared with the levels at the same time of the day at 25 and 90 days. This increase persisted in autumn and spring. The 24-hr pattern in hypothalamic melatonin was the inverse of that in the pineal, with the levels at noon higher than those at midnight. This pattern was detectable at 25 days of age although the difference in melatonin between 12:00 and 24:00 hr was not great. The day/night difference was prominent by 55-60 days of age and disappeared by adulthood (90 days). This 24-hr pattern was similar in spring and autumn in the three ages studied. Although in the 55-60-day-old group the melatonin ratio (noon/midnight) was the same in autumn and spring, the absolute levels of melatonin in spring were significantly lower. The findings are consistent with the general concept of a modulatory role of melatonin in control of hypothalamo-hypophyseal GnRH and gonadotropin function, and the timing of the developmental maturation of this neuroendocrine axis. Demonstration of the mechanism of melatonin's action at the hypothalamic level will be facilitated by further definition of quantitative developmental changes.

Effects of melatonin on adrenomedullary dopamine-beta-hydroxylase activity in golden hamsters: evidence for pineal and dose dependencies.

Pineal influence in the control of adrenomedullary function in golden hamsters was investigated by examining changes in adrenal dopamine-beta-hydroxylase (DBH) activity following pinealectomy, either alone or in combination with melatonin administration. Adult males acclimated to an LD 14:10 photoperiod were distributed in five experimental groups: intact controls (NO), sham-pinealectomized (S), sham-pinealectomized with black plastic shielding of the pineal region, pinealectomized (PX), and pinealectomized with the operated region shielded. Animals representing all of these groups were injected (between L11 and L11.75) with either vehicle, or a low dose (25 micrograms) or a high dose (2,500 micrograms) of melatonin daily for 28 days, after which they were killed, and the adrenals were collected for assay of DBH activity by means of a sensitive radioenzymatic method. We found that PX + vehicle led to increased (P less than .05) adrenal DBH activity in comparison with either NO or S groups; daily 25 micrograms of melatonin resulted in lowered DBH activity in the NO group when compared with NO + vehicle (P less than .001) or S + vehicle (P less than .001) groups; PX + 25 micrograms melatonin reversed the action of 25 micrograms melatonin in the NO + 25 micrograms group; 2,500 micrograms melatonin was without effect on adrenal DBH in any of the injected surgical groups. These results show an inhibitory pineal influence on adrenal DBH activity, and that this was dose dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Increases in volume, fluid content, and lens weight of eyes following systemic administration of melatonin.

Melatonin's effects were studied in male golden hamsters (Mesocricetus auratus) distributed among five surgical groups (nonoperated, sham-pinealectomized, sham-pinealectomized plus black plastic shielding of the pineal region, pinealectomized, and pinealectomized plus black plastic shielding of the pineal region) and three injection groups (vehicle only, 25 micrograms melatonin, and 2,500 micrograms melatonin). Injections (s.c.) were daily for 28 d at L11 to L11.75 in a (light:dark) L:D 14:10 artificial photoperiod. Animals (N = 112) were killed and dissected on the day after the last injection (at 55-65 d of age). None of the surgical procedures affected weights of eyes or their parts, nor did they influence the effects of administered melatonin on the eyes. Melatonin caused an increase in absolute and relative eye weight and an increase in fluid content of intraocular space. The magnitudes of these effects were positively related to melatonin dose. These same eyes had a progressively lower weight of nonlenticular tissues with low to high doses of melatonin, probably in relation to greater fluid content, and suspected increase in intraocular pressure. Lens wet and dry weights were significantly greater in animals receiving melatonin, but only at the high dose. These actions of melatonin are likely to be direct and are shown to not require the presence of the pineal. Experiments of other designs are suggested in order to determine whether the effects of the low, near physiological, dose of melatonin represent physiological actions of endogenous melatonin, synthesized and released within the eye. However, effects of large doses of melatonin on the eye are still noteworthy in relation to interpretation of experiments employing such dosages, and of disease states involving changes in intraocular pressure.

Stimulatory effects of melatonin on ependymal epithelium of choroid plexuses in golden hamsters.

Ependymal epithelial cells from the choroid plexuses (CPs) separately of lateral (I + II), IIIrd and IVth ventricles of male Golden Hamsters were studied by electron microscopy and morphometry. The 16 hamsters were distributed between three injections groups: vehicle only, 25 micrograms and 2500 micrograms melatonin (M) by subcutaneous injection daily at L11 to L11.75 in a LD 14:10 daily photoperiod. After 28 consecutive daily injections, animals were killed and the CPs were dissected, fixed and prepared for electronmicroscopy. Thirteen measures of the CP ependymal cells were made, by planimetry, morphometry or direct counting or linear measurement on the EM prints. Effects of melatonin occurred only on the cells from the lateral ventricles. Here M at high dosage caused cell swelling (averaging 50% increase in area), and other cellular changes were graded in relation to M dosage. These were increased (to 26%) in mitochondrial area per cell, and increased (to 50%) in length of apical microvilli. Since in other work the latter form a major locus of ouabain-sensitive Na+, K+-ATPase, it is suggested that M may possibly have a stimulatory effects on transport and related CSF secretory activities by these cells.

Adrenal and hypothalamic dopamine-beta-hydroxylase activity in the hamster: evidence for species-distinctive day-night and post-pinealectomy differences in activity.

Dopamine-beta-hydroxylase (DBH) activities were measured in the adrenal gland and hypothalamus of the golden hamster (Mesocricetus auratus) by means of a sensitive radioenzymatic method. Groups of unoperated control (C), sham pinealectomized (SPX), and pinealectomized (PX) adult males were sacrificed shortly before (09:00 h) and after (11:00 h) the onset of darkness of the daily photoperiod of LD 12:12 (lights on 22:00 h to 10:00 h). In neither adrenal gland nor the hypothalamus were there any day-night changes in the C groups. Pinealectomy led to no significant differences in hypothalamic DBH activity either in light or in dark. However, intracranial surgery resulted in increased DBH activity in the adrenal gland, evident in both light and dark. Although this increase was greatest in the PX groups, intracranial surgery in SPX groups also showed a trend of increase in adrenal DBH, evident even one month after surgery. Such an increase, however, was not found in hypothalamic DBH in the same SPX groups. In both the adrenal gland and the hypothalamus, copper-sensitive endogenous inhibitors of DBH did not show any notable change in concentration, either in relation to the daily light-to-dark change or as a consequence of intracranial surgery. These and related results suggest that important interspecies variations exist in DBH activities in relation to diurnal changes, and also in control or modulation by pineal and other factors.

Effect of methionine, glycine and serine on serine hydroxymethyltransferase activity in rat glioma and human neuroblastoma cells.

Human neuroblastoma SK-N-SH-SY5Y (5Y) and rat glioma (C6) cells were cultured with supplemental methionine, glycine, or serine for three to six days. Serine hydroxmethyltransferase (SHMT: L-serine: tetrahydrofolate 5, 10-hydroxymethyltransferase, EC 2.12.1) was assayed radiometrically in whole cell homogenates, crude supernatant fractions and crude particulate fractions. No significant changes in specific activity or cellular morphology were noted at methionine, glycine, or serine concentrations up to 16 mM. Serine concentrations of 20 and 40 mM led to significantly lower gliomal enzyme specific activities. This activity was unevenly distributed between soluble and particulate fractions, with 190 and 398 nmoles of HCHO formed per mg of protein per hour, respectively. Growth stage and time of incubation were major determinants of enzyme specific activity. C6 cells' specific activity rose slowly with increasing time in culture until cellular confluence. At this time there was a pronounced elevation in specific activity, occurring more rapidly in cells grown in 1.2 mM methionine. Intracellular amino acid analysis of C6 cells demonstrated a significant rise in methionine after four days in media containing 0.2 mM methionine. No appreciable diminution in the intracellular levels of glycine or serine occurred following incubation in excess methionine. It is concluded that SHMT-specific activity in C6 and 5Y cells is not regulated by glycine, serine, or methionine levels and that high concentrations of these amino acids (> 30 mM) are not detrimental to these cells derived from the CNS.

Androgens and brain differentiation. Effects of testosterone on protein synthesis in neonatal rat hypothalamic and cerebral slices.

Protein synthetic activity of anterior hypothalamic and neocortical brain slices from androgenized neonatal female rats was studied in vitro in an attempt to clarify the mechanism of action of testosterone on the developing hypothalamus. The short-term effects of testosterone propionate injections of the animals on the protein synthetic activity of the slices were assayed by means of incorporation of 14C-leucine into acid-insoluble protein. Although a high dose (1 mg/animal) of testosterone propionate stimulated increased incorporation in neocortical and anterior hypothalamic slices, a lower dose and other approaches failed to demonstrate a stimulation regionally specific to the anterior hypothalamus. It is concluded that if testosterone has specific stimulatory effects on protein synthesis in the anterior hypothalamus of neonatally androgenized rats, these are not readily demonstrable by methods used with other steroid target tissues.

Hypothalamic dopamine-beta-hydroxylase activity: fluctuations with time of day and their modifications by intracranial surgery, adrenalectomy, and pinealectomy.

Hypothalamic and plasma dopamine-beta-hydroxylase [DBH, EC 1.14.2.1] activites were measured by a coupled radioenzymatic method. Animals representing five experimental groups [intact controls, adrenalectomized, pinealectomized, adrenalectomized pinealectomized, doubly sham-operated] were killed and sampled at 8 times through the 24-hr daily cycle, 15 days postoperation, and at 50-52 days of age. Hypothalamic DBH in intact control animals had statistically significant fluctuations in relation to time of day. These changes were lost or dampened in groups that had had intracranial surgery and were characteristically shifted by adrenalectomy, either alone or with pinealectomy. Plasma DBH fluctuations in the same animals resembled those in hypothalamus in some features (e.g., peak near mid-dark; shift in daily maxima and minima after adrenalectomy) and differed in others [e.g., no effect of intracranial surgery or of sham operation; adrenalectomized pinealectomized animals resembled the solely pinealectomized]. Although temporal patterns in hypothalamic DBH activity thus differed in the experimental animal groups, the daily means of hypothalamic DBH activity were similar.

Hypothalamic and suprachiasmatic uptake of serotonin in vitro: twenty-four-hour changes in male and proestrous female rats.

Twenty-four-hour changes in the in vitro serotonin (5-HT) uptake capacity of hypothalamic homogenates and of "Vibratome" slices of the suprachiasmatic nuclear region (SNR) of the hypothalamus were studied in 60-90-day-old Holtzman (albino) rats. Animals acclimated to artificially illuminated (fluorescent, LD 12:12) and temperature controlled (22 +/- 2 C) rooms were killed 6% each of 8 time points. Synaptosomal fractions from homogenates of whole hypothalamus, and slices of the SNR were incubated for 20 min in Krebs-Henseleit buffer with [3H]5-HT. Males showed a single daily peak in SNR uptake at the start of darkness, and a minimum near the onset of light, while a more complex pattern containing 3 peaks and minima occurred in uptake by hypothalamic homogenates. Proestrous females showed a single high amplitude peak SNR uptake during the critical period, just prior to the plasma LH peak determined in the same animals by radioimmunoassay. It is suggested that this short-term and 4-fold increase in SNR uptake of 5-HT may serve to limit free 5-HT and its inhibitory or other effects on the gonadotropin release hormone system and thereby on LH release and ovulation.

Effects of continuous light and darkness, and of pinealectomy, adrenalectomy and gonadectomy on uptake of 3H-serotonin by the suprachiasmatic nuclear region of male rats.

Rats exposed to continuous light show no significant differences in serotonin (5-HT) uptake by the suprachiasmatic nuclear region (SNR) at 10.00 and 22.00 L, while in continuous darkness, the uptake at 10.00 D is significantly higher than uptake at 22.00 D. In consideration of the role of the suprachiasmatic nucleus in light-mediated control of neuroendocrine activity, this suggests endogenous generation or control of this uptake rhythm. An effect of sham-pinealectomy in decreasing 5-HT uptake was found, but cannot be fully interpreted at this time. The lack of effect of castration or adrenalectomy suggests that these endocrines have little if any direct effect on 5-HT mechanisms in the SNR.

The effects of castration on histamine levels and 24-hour rhythm in the male rat hypothalamus.

Holtzman male rats were castrated or sham-operated at 22 days of age and raised in a 12 h light: 12 h dark illumination cycle. At age 63 days they were sacrificed by decapitation at six different times during the light:dark (L:D) cycle, and hypothalamic histamine (H) concentrations were measured using a modified single isotope-enzyme microassay. Hypothalamic H was significantly elevated in the castrated rats at all but two of the six times sampled during the L:D cycle, when compared with the sham-operated controls. Both surgical groups had similar 24-h rhythms of hypothalamic H concentrations, with the peak concentration occurring during the light phase followed by a rapid drop to the minimum 2-3 h later. However, the castrated rats appeared to attain both maximal and minimal concentrations somewhat earlier in the day. These results provide circumstantial evidence that hypothalamic H may have a role in the hypothalamo-hypophyseal-gonadal axis in the male rat.

Hypothalamic 24-hour rhythms in histamine, histidine, decarboxylase and histamine-N-methyltransferase.

Twenty-four hour rhythms of histamine (H), its biosynthetic enzyme, L-histidine decarboxylase (EC 4.1.1.22, HD) and its inactivation enzyme, histamine N-methyltransferase (EC 2.1.1.8, HMT) were assayed in the 63-day-old male rat hypothalamus using a modified single isotopeenzyme microassay. H was found to have a 24-h rhythm with a maximum concentration at 11:00 h (lights on 10:00-22:00 h) and a minimum at 23:00 h. In vitro HD and HMT activities were also found to have 24-h rhythms with peak activites in both occurring at 04:00 h. HMT also had a broad secondary peak throughout the day, whereas HD activity during the day was dominated by a mid-light trough at 16:00 h. These rhythms are considered significant for evaluation of the control and physiological role of histamine in the mammalian hypothalamus.