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Journal of Neuroscience, Vol 14, 3945-3957, Copyright © 1994 by Society for Neuroscience

ARTICLE

An in vitro model for the effects of estrogen on neurons employing estrogen receptor-transfected PC12 cells

RH Lustig, P Hua, W Yu, FJ Ahmad and PW Baas
Department of Pediatrics, University of Wisconsin, Madison 53792.

Estrogen alters neurite outgrowth, neuritic spine development, and synaptogenesis in estrogen-responsive areas of the rat brain. However, examination of the specific effects of estrogen on neurons in vivo has been difficult. An in vitro model for the effects of estrogen on neurons was developed, using the PC12 rat pheochromocytoma cell line. Wild-type 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, screened for incorporation of vector and expression of ER mRNA and protein, and analyzed for morphologic responses to estrogen. PC12-WT, NEO9 (ER-negative), and SER8 (ER-positive) cells exposed to 100 ng/ml NGF exhibited dose-responsive neurite outgrowth within 2 d by light microscopy (LM). Coadministration of 10(-10) to 10(- 9) M estradiol (E2) had minimal effects on neurite outgrowth, neuritic spine development, or interneuritic connections in NEO9 or PC12-WT cells, but in SER8 cells E2 led to additive and dose-dependent increases in neurite outgrowth, spine development, and interneuritic connectivity. Coincubation of SER8 cells with E2 and the antiestrogen ICI 164,384 negated estrogenic effects on spine development and interneuritic connectivity. At the electron microscopic (EM) level, intercellular abutments of NEO9 or PC12-WT cells contained few and rudimentary gap junctions, with no increase by E2. However, SER8 cells exhibited augmented basal frequencies of gap junctions that increased with E2 incubation. Microinjection of Lucifer yellow into PC12-WT and NEO9 cells demonstrated low frequencies of dye coupling and no change with E2, but SER8 cells demonstrated increased dye-coupling frequency with E2 coincubation. The results suggest that SER8 cells recapitulate estrogen effects on neurons in vivo. Estrogen appears to induce an inherent neural morphologic program in estrogen receptor (ER)- containing cells. These three cell lines provide a unique in vitro system for studying mechanisms of estrogen-neuron interactions.

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