Sex hormone dose escalation for treating abnormal sleep in ovariectomized rats: in vitro GABA synthesis in sleep-related brain areas

Abstract

No data in the literature have evaluated sex hormone dose escalation for treating abnormal sleep of ovariectomized rats—nor studies on the role of sex hormones in GABA synthesis of rats' sleep-related areas. The main aim of this study was to determine the maximum tolerated dose (MTD) of estradiol (ET), progesterone (PT), and the mixture of both (EPT) to restore normal sleep in a model of menopause in rats. The second purpose was to describe the in vitro activity of glutamate decarboxylase (GAD) in sleep-related brain areas in the presence or absence of sex hormones. A weekly dose-escalation design of ET, PT, or EPT was implemented in ovariectomized rats (six per group). Dose escalation continued until the dose at which 100% of the rats exhibited a state of “complete somnolence.” Doses that were not toxic or did not show side effects were considered. For in vitro experiments, sleep-related brain areas were separated and incubated with radiolabeled glutamate. Estradiol (17β-E2), progesterone (P), and pyridoxal phosphate (PLP) were added to this assay, and GAD activity was determined. Under the same conditions, a second test was carried out, but the P antagonist RU486 was added to assess the role of P in GAD activity. Ovariectomy increased periodic awakenings compared to those determined for the SHAM group. The EPT for ovariectomized rats was very effective by the fifth week in decreasing arousal and achieving a similar sleep behavior to the SHAM-control group. Rats tolerated the ET, PT, and EPT well to the maximum planned dose (0.66 mg/kg and 4.4 mg/kg, respectively). No lethal events occurred; the MTD was reached. The in vitro studies indicated that the presence of 17β-E2 plus P in the assay triggered the activity of isotype 65 GAD in all the studied brain areas. RU486 in the incubation medium blocked such activity; however, the action of isotype 67 GAD was not blocked by RU486. A dose-escalation model was determined; the MTD coincided with the maximum dose of ET and PT used. However, the EPT combination restored normal sleep in the menopause model compared to the SHAMs without toxic effects. The in vitro model demonstrated that 17β-E2 plus P presence in the assay increased the activity of GAD65 in the studied brain tissues.