Amygdala neurons in vitro: neurite growth and effects of estradiol

Dissociated cell cultures from embryonic rat medial amygdala were studied using sequential photography and immunocytochemical staining for cytoskeletal proteins and substance P (sP). Cultures were seeded with cells taken from fetuses grouped by sex; experimental cultures were raised in medium containing 17-beta-estradiol (E2). Forty-eight hours after plating a few neurons begin to define their morphological polarity by the differentiation of an axon-like process; at 5 days in vitro (DIV) almost all neurons had developed an axon. Tapering, daughter branch ratio and branch power coefficient coincided with identification of dendrites which could be confirmed by retrospective analysis of immunocytochemically stained cultures: at 5 DIV MAP-2 was restricted to dendrites whereas Tau immunoreactivity was differentially localized with a clear predominance in the axon. At 21 DIV neuronal shape parameters were strikingly like those of amygdaloid neurons in vivo. It was demonstrated in living neurons that E2 increased total dendritic length and that this is due to increased ramification of third or higher order dendritic segments whose individual lengths are not different from controls. Densitometric measurement of MAP-2 stained neurons showed a highly significant increase of immunoreactive material in cells grown in the presence of E2; readings for alpha-tubulin were not different between controls and E2 treated cultures. The effect of E2 on dendritic length was just as manifest in sP-positive as in sP-negative neurons. No sexual differences in morphological parameters, growth characteristics or effects of E2 were found in neurons taken from female fetuses versus neurons from male fetuses. The significance of these results for the generation of sexual differences in the amygdala in vivo is discussed and contrasted with reported results on the effects of E2 in cultures of different neural regions.