ESTROGEN EFFECTS ON SEPTAL CHOLINERGIC NEURONS IN CULTURE





J. Stoll, C. Martin, C. Hoelting

Dept. Pharmaceutical Sciences, Texas Tech Univ HSC, School of Pharmacy, 1300 Coulter, Amarillo, TX 79106



The purpose of this study was to investigate the actions of estrogen in an in vitro model system of cultured basal forebrain neurons. Previous work by a number of investigators indicated that estrogen regulates the function of basal forebrain cholinergic neurons in vivo. Our goal is to understand the mechanism of these actions by investigating them in an in vitro culture system. Basal forebrain/septum was dissected from fetal rats (days 16-17 of gestation), dissociated into single cells, and grown in serum-free medium (DMEM:Neurobasal [1:1] + B27 supplements + 50 ng/ml nerve growth factor). Cholinergic neurons represent approximately 1% of the total cell population as assessed by immunostaining for a specific cholinergic marker, the vesicular acetylcholine transporter. In cultures containing 0.1 or 1 nM beta-estradiol, the fraction of cholinergic neurons increased 50%. A similar increase was observed in cultures treated with the phytoestrogen daidzein (0.4 mcM), but not t! he related phytoestrogen, genistein. These results suggest that estrogen and certain related compounds are trophic and enhance the survival of basal forebrain cholinergic neurons in vitro. The effect of estrogen on high affinity choline transport (HACT), the rate-limiting step in acetylcholine synthesis, was also determined. HACT was measured by determining the amount of [3H]choline uptake sensitive to 0.3 mcM hemicholinium-3. This concentration was chosen based on initial experiments in which hemicholinium-3 inhibited HACT with a Ki of 2.3 nM. The ubiquitous low affinity choline transporter was inhibited by hemicholinium-3 with a Ki of 26 mcM. HACT was variable between 0-28% of total choline uptake in several different culture preparations. Estradiol treatment was shown to increase HACT in those cultures containing a significant amount of high affinity uptake. Results of one experiment showed an increase from 29 dpm/mcg protein (19% total) in control cells to 41 dpm/mcg pro! tein (24% total) in cells treated with 25 nM estradiol. These results provide preliminary evidence that estrogen enhances the survival and function of cholinergic neurons in this model system and open the door to investigations into the mechanism of the effect. These results may lead to new therapeutic approaches in Alzheimer's disease and in aging in which basal forebrain cholinergic neurons degenerate.




Key words: acetylcholine, neurodegeneration, phytoestrogen, choline transport







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