Library

Notes: Abstract— Ovariectomized female rats were treated daily with oestradiol-17β benzoate for intervals up to one week and enzyme activities were measured in the pituitary and various brain regions. Brain regions were selected for study on the basis of their previously demonstrated content of putative oestradiol receptor sites. (1) Pituitary showed oestrogen-dependent increases in glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and lactic dehydrogenase (LDH), and no change in NADP+-dependent isocitric dehydrogenase (ICDH), NADP+-dependent malic dehydrogenase (MDH) or hexokinase (HK). MDH and ICDH were elevated in whole hypothalamus. Enzyme activities did not change significantly in whole amygdala, cerebral cortex, or hippocampus. (2) Sub-regions of the preoptic area, hypothalamus and amygdala were dissected to obtain more highly concentrated populations of cells containing putative oestrogen receptor sites. In the basomedial sub-region of hypothalamus, activities of MDH, ICDH and G6PDH were elevated by oestrogen treatment. In the corticomedial sub-region of amygdala, MDH and ICDH were elevated by oestrogen treatment. No change was observed in any of the six enzymes in medial preoptic area. (3) Increases in enzyme activities were related to the total in vivo dose of oestradiol benzoate given. (4) Hypophysectomy or adrenalectomy did not prevent the enzymatic responses to oestrogen. (S) Oestrogen added directly to the enzyme incubation medium did not change enzyme activities. (6) Weight loss in ovariectomized rats due to reduced food intake did not increase enzyme activities. (7) In the pituitary, good correlation was obtained between the known receptor binding properties of various oestrogenic and non-oestrogenic steroids and the elevation in G6PDH activity. The results indicate that oestradiol acts directly to cause changes in activities of some brain and pituitary enzymes. The possibility is discussed that these changes may result from oestrogen interaction with putative receptor sites found in pituitary and certain brain regions.

Notes: After injection of the tritiated RNA precursors [3H]guanosine, [3H]uridine or [3H]orotic acid into the eye of goldfish, labelled TCA-soluble material and RNA appeared to be axonally transported to the contralateral optic tectum. From the time courses of arrival in the tectum,‘average’rates of transport of 6 mm/day for the soluble material and 1·7 mm/day for the RNA were calculated. If the optic nerve was cut after the transported material had arrived in the tectum, about 60 per cent of the TCA-soluble material disappeared by 7 days after the cut, but almost none of the RNA. After a further 8- to 13-day period, the TCA-soluble material had declined by a further 50 per cent from the 7-day value, but the RNA by only 20 per cent. Thus, relatively little RNA was lost when the optic axons degenerated, an observation which suggested that the RNA might be extra-axonal. However, if the optic nerve was crushed before the arrival of the transported material, RNA did not appear in the tectum until the regenerating optic nerve fibres arrived. Therefore, the presence of RNA must be dependent on intact nerve fibres. Moreover, in the earliest stages of regeneration the proportion of transported RNA to TCA-soluble material was considerably higher than normal, suggesting that the regenerating fibres arrived in the tectum already carrying RNA. This implies that the RNA itself was transported in the optic fibres.

Notes: —Cell nuclei were isolated from four regions of the brains of ovariectomized female rats 2 hr after the injection of [3H]oestradiol. By light microscopy, the nuclear pellets contained highly purified nuclei of neuronal and glial cells with little cytoplasmic contamination. Tritium was concentrated in cell nuclei from the preoptic-hypothalamic area, to a lesser extent in nuclei from the amygdaloid region and hippocampus, and least of all in cerebral cortical nuclei. In comparison with whole homogenates (= 1-0), the nuclear concentrations of radioactivity were 12·9, 4·7, 1·9 and 0·8, respectively. Approximately 40 per cent of the radioactivity in homogenates of the preoptic-hypothalamic area was present in cell nuclei, and upon TLC more than 85 per cent of the radioactive material in the nuclei exhibited the RF of oestradiol-17β.Pretreatment of ovariectomized females with 1 mg of unlabelled oestradiol 30 min before the injection of labelled hormone abolished the nuclear uptake of [3H]oestradiol in all four regions of the brain. A concurrent injection of 10 μg of unlabelled oestradiol-17β significantly reduced nuclear uptake, while a similar injection of testosterone or oestradiol-17α had no significant effect. One mg of oestradiol-17α, but not testosterone, did reduce nuclear uptake.The retention of [3H]oestradiol by the preoptic-hypothalamic area decreased exponentially in the tissue from 30 min to 4 h after an intraperitoneal injection; however, nuclear binding reached a peak at 1-2 h and still showed high retention at 4 h. These results, together with observations in other laboratories of morphological changes induced by oestrogens, establish that certain regions of the brain are bona fide targets for the action of oestradiol.

Notes: —After injection of [3H]guanosine or [3H]uridine into the eye of goldfish, labelled acid-soluble radioactivity and RNA appeared in the contralateral optic tectum. When 0·1 μg actinomycin-D was injected into the eye 4 h before the precursor, the labelled RNA in the retina by 18 h after the injection was only 23 per cent of normal, but the acid-soluble radioactivity in the retina and the small amount of labelled acid-soluble material conveyed to the tectum were not significantly affected; by 15–20 days after the injection the acid-soluble radioactivity in the retina was reduced and the amount of labelled material conveyed to the tectum, including both RNA and acid-soluble fractions, was less than normal.When the actinomycin was injected at various times before or after the precursor and measurements were made 6 days later, it was found that the amount of labelled RNA conveyed to the tectum was maximally decreased if the inhibitor was given simultaneously with or up to 4 h before the precursor, whereas the amount of RNA was normal if the incorporation of the precursor had been allowed to proceed for 12 h before the inhibitor was given. This result would be consistent with the view that much of the RNA conveyed to the tectum had been synthesized in the retina within 12 h of the injection of the precursor, and had then presumably been axonally transported in the optic nerve to the tectum. However, since the acid-soluble material conveyed to the tectum was also reduced as a result of the actinomycin treatment, the results of these experiments with actinomycin do not unequivocally rule out the possibility that the RNA appearing in the tectum had been locally synthesized from the axonally transported acid-soluble material. In the retina, both the labelled RNA and acid-soluble fractions were reduced, to about 15 and 60 per cent of normal, respectively, without any relationship to the time between the injection of inhibitor and precursor. The discrepancy between the effects of the labelling of the retina and the labelling of material conveyed to the tectum could be correlated with the fact that the actinomycin caused severe damage to the retinal receptor cells, while leaving the ganglion cells relatively intact. The more pronounced effect of actinomycin on the receptor cells could in turn be correlated with the fact that these cells had a higher rate of RNA synthesis than the ganglion cells. This was demonstrated autoradiographically by the higher rate of incorporation of [3H]uridine into the receptor cells.Intracranial injection of actinomycin did not affect significantly the amount of labelled RNA conveyed to the tectum, which would argue against the local synthesis of this RNA. It is not certain, however, that the actinomycin penetrated deeply enough into the tectum to be effective.

Notes: Abstract— Cell nuclei were isolated in yields ranging from 38 to 61 per cent from six anatomically defined brain regions of the albino rat. To provide basic information for further studies of altered genomic activity in brain cell nuclei, various properties of these isolated nuclei were measured, including counts of their number, estimates of the distribution of sizes, amounts of RNA, DNA and protein, and endogenous RNA polymerase activity. DNA content per nucleus approximated the accepted value of 6 pg per diploid set of chromosomes. Distributions of nuclear size showed a sensitivity to the concentration of divalent cation, with a shift toward larger nuclear diameters as the Mg concentration was reduced. Cell nuclei from hippocampus, hypothalamus-preoptic region, cerebral cortex, amygdala and midbrain plus brainstem were generally similar in yield, distribution of size, and RNA, DNA and protein content. Cell nuclei from cerebellum differed from those of other brain regions, in all of these parameters. The cerebellum contained a high content of DNA and had an enormous number (8 × 108 per g wet wt.) of cell nuclei of predominantly very small size and characterized by lower ratios of RNA, histones and non-histone protein to DNA and lower endogenous activity of RNA polymerase than nuclei from other brain structures. These properties correlated well with properties of cerebellar tissue, namely, high content of small granule neurons and low ratio of RNA to DNA, and suggest that the small cerebellar nuclei may have relatively inactive genomes. The relationship of ‘large’ and ‘small’ cell nuclei to cell types in the brain is discussed.

Notes: The gonadal steroids estradiol and progesterone have previously been shown to modulate the specific binding of the GABAA agonist, [3H]muscimol, in the CA1 region of the hippocampus, the ventromedial nucleus of the hypothalamus and the midbrain central gray of ovariectomized female rats. In this report we show a sex difference in the level of binding in the very caudal ventromedial nucleus of the hypothalamus. In contrast to females, there is no steroid modulation of [3H]muscimol binding in the ventromedial nucleus of the hypothalamus and midbrain central gray of males. These effects may be functionally related to GABAergic control of female sexual behavior. In contrast, steroid modulation of [3H]muscimol binding in the CA1 region of the hippocampus occurred to the same degree in males and females, and there was no difference in the level of binding in any region of the hippocampus between gonadectomized males and females.Incubation of brain slices with progesterone or its metabolite 5α-3α-pregnanolone dissolved in ethanol, produced a significant increase in [3H]muscimol binding in most brain regions as compared to control brain slices treated with ethanol alone. Moreover, there was also a marked increase in [3H]muscimol binding in all brain areas in the control condition which contained 100 mM ethanol, as compared to brain slices not preincubated with ethanol. The increase in binding after in vitro treatment with either progesterone or 5α-3α-pregnanolone is notably different from that seen after progesterone given in vivo 4 h prior to assay in that it is not site-specific, does not depend on prior treatment with estradiol and shows no sex difference. These results suggest different mechanisms for progesterone effects on the GABAA receptor when administered in vivo as compared to in vitro.

Notes: Abstract— Administration of oestrogen (oestradiol-17β or oestradiol-17β-benzoate) to ovariectomized (OVX) rats for 1–4 weeks results in an approx 30% decrease in the activity of monoamine oxidase (MAO) in the basomedial-hypothalamus (BM-Hyp) and corticomedial-amygdala (CM-Amy) but not in cerebral cortex. Further investigation shows that (1) decreased MAO activity in the BM-Hyp and CM-Amy occurs only in Type A MAO (serotonin as substrate) and does not occur in Type B MAO (phenylethylamine as substrate); (2) decreased MAO activity does not occur when a single large dose of oestrogen is given i. v. or when homogenates from oestrogen treated rats are mixed with homogenates from OVX rats suggesting that direct enzyme inhibition is not responsible for the change in activity; (3) oestrogen administration to OVX rats increases the rate constant of degradation for MAO in BM-Hyp and CM-Amy but not in cerebral cortex as determined in turnover studies using pargyline, an irreversible inhibitor of MAO. The increased rate of degradation results in shorter half lives (t 1/2) for MAO in the BM-Hyp and CM-Amy of oestrogen treated rats. In OVX rats the t 1/2 is 9.8 days in BM-Hyp and 12.7 days in CM-Amy. Oestrogen administration results in a t 1/2 of 7.6 days in BM-Hyp and 7.8 days in CM-Amy. The possible relationship between oestrogen dependent decreased MAO activity and estrogen dependent lordosis behavior is discussed.