AN IN-VITRO MODEL FOR THE EFFECTS OF ESTROGEN ON NEURONS EMPLOYING ESTROGEN RECEPTOR-TRANSFECTED PC12 CELLS

Estrogen alters neurite outgrowth, neuritic spine development, and syn aptogenesis in estrogen-responsive areas of the rat brain. However, ex amination of the specific effects of estrogen on neurons in vivo has b een difficult. An in vitro model for the effects of estrogen on neuron s was developed, using the PC12 rat pheochromocytoma cell line. Wild-t ype cells (PC12-WT) were stably transfected either with an expression vector coding for the full-length cDNA for the human estrogen receptor (hER), or with a control vector. Resultant clones were isolated, scre ened for incorporation of vector and expression of ER mRNA and protein , and analyzed for morphologic responses to estrogen. PC12-WT, NEO9 (E R-negative), and SER8 (ER-positive) cells exposed to 100 ng/ml NGF exh ibited dose-responsive neurite outgrowth within 2 d by light microscop y (LM). Coadministration of 10(-10) to 10(-9) M estradiol (E(2)) had m inimal effects on neurite outgrowth, neuritic spine development, or in terneuritic connections in NEO9 or PC12-WT cells, but in SER8 cells E( 2) led to additive and dose-dependent increases in neurite outgrowth, spine development, and interneuritic connectivity. Coincubation of SER 8 cells with E(2) and the antiestrogen ICI 164,384 negated estrogenic effects on spine development and interneuritic connectivity. At the el ectron microscopic (EM) level, intercellular abutments of NEO9 or PC12 -WT cells contained few and rudimentary gap junctions, with no increas e by E(2). However, SER8 cells exhibited augmented basal frequencies o f gap junctions that increased with E(2) incubation. Microinjection of Lucifer yellow into PC12-WT and NEO9 cells demonstrated low frequenci es of dye coupling and no change with E(2), but SER8 cells demonstrate d increased dye-coupling frequency with E(2) coincubation. The results suggest that SER8 cells recapitulate estrogen effects on neurons in v ivo. Estrogen appears to induce an inherent neural morphologic program in estrogen receptor (ER)-containing cells. These three cell lines pr ovide a unique in vitro system for studying mechanisms of estrogen-neu ron interactions.