Endocrine Abstracts

We determined in vitro activity of 17β-estradiol and or progesterone on glutamate conversion to GABA in different GABAergic regions from the brain of castrated and non-castrated rats. The glutamate decarboxylase catalyzes this conversion; there are two different isotypes of glutamate decarboxylase, GAD₆₅ and GAD₆₇. Whereas GAD₆₅ is a membrane enzyme, GAD₆₇ is present in the supernatant obtained after centrifuging to 14, 000 g. We treated three different groups of ovariectomized rats with increasing week-dose of 17β-estradiol, progesterone, and the combination of both for five weeks. We maintained one group of the ovariectomized rats as a control. After five weeks of treatment, we separated the rat-brain. We dissect the brain regions which have previously been related to the sleep process: mammillary bodies, locus coeruleus, substantia nigra, and the ascending reticular activating system. After homogenates and centrifuge these tissues, we incubating the membrane fraction and supernatant separately with tritiated-glutamate a pyridoxal phosphate. We assessed the decarboxylation of glutamate for the formation of a Schiff base, which was purified by thin-layer chromatography. We identified this Schiff base by its fluoresces at λ 302 nm. We quantify this conversion by counting the radioactivity present in the fluorescence zone. Results indicated that 17β-estradiol-treatment increased the GAD₆₅ and GAD₆₇ activity in all the studied brain-regions compared with those of the ovariectomized (vehicle-treated) control. In contrast, progesterone increased GAD₆₅ and GAD₆₇ activity only in the ascending reticular activating system and locus coeruleus. Furthermore, 17β-estradiol plus progesterone-treatment also increased GABA synthesis in the ascending reticular activating system and locus coeruleus compared to the vehicle-treated control. However, the ovariectomized (vehicle-treated) control showed only GAD₆₅ and GAD₆₇ activity in the mammillary bodies and substantia nigra. The brain GABAergic structures studied are involved in the process of sleep. Therefore the increase of the GABA formation induced by 17β-estradiol is responsible for maintaining sleeping in rats. However, the progesterone role seems to be a regulator of this process because the locus coeruleous and the ascending reticular activation system are responsible for inducing the sleep-wake rhythm.