TIME-LAPSE STUDY OF NEURITE GROWTH IN HYPOTHALAMIC DISSOCIATED NEURONS IN CULTURE - SEX-DIFFERENCES AND ESTROGEN EFFECTS

Cultures of dissociated hypothalamic cells taken from rat fetuses of 19 days of gestation were studied using time-lapse recording and sequential microphotography from 1 to 5 days in vitro (DIV) and at 7 and 21 DIV. Cultures were seeded with cells taken from fetuses grouped by sex or sexually mixed; experimental cultures were raised in medium containing 17-beta-estradiol 100 nM (E2). Cells were plated on poly-D-lysine-coated coverslips at a culture density of approximately 4,000 cells/cm2. Immunocytochemistry of cell cultures was performed using a Tau monoclonal antibody (clone Tau-1 PC1C6) and a monoclonal antibody against MAP-2 (clone AP-20). Cells started to produce lamellipodia and neuritic processes approximately 4 hr after plating. Forty-eight hours later a few neurons had defined their morphological polarity by the differentiation of an axon-like process that grows faster than the others; at 5 DIV almost all neurons had defined their axons. At this time, monoclonal antibody against MAP-2 clearly stained soma and dendrites, but not axons. Tau immunoreactivity (lots CCA101 and CCA101N from Boeringher Mannheim) was differentially distributed, with a clear predominance in axon and soma. Results on the morphometric analysis of control and E2 treated neurons provide direct evidence for the existence of sex related differences in the neurite outgrowth response of hypothalamic neurons, since cultured neurons taken from female fetuses differentiated axons later and had fewer primary neurites and shorter dendrites than neurons taken from male fetuses or sexually mixed cultures. Also, it was demonstrated in living neurons that E2 effectively enhances outgrowth and elongation in axons. The frequency distribution curves of axonal length for control and E2 treated cultures was unimodal, suggesting that the effect of E2 was a uniform increase in the axonal length of all neurons. The structural differences between neurons from both sexes and the changes induced by E2 may contribute to explain the differences in brain function found between the sexes.