Amygdala neurons in vitro: Neurite growth and effects of estradiol

Abstract

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.

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system

“…In aged animals and in different lesion paradigms, the treatment with estradiol enhances neuronal survival and recovery (Tanzer and Jones, 1997;Callier et al, 2000;Wise et al, 2000;Garcia-Segura et al, 2001). In culture, estradiol protects neurons against excitatory amino acid and ␤-amyloid peptide toxicity, oxidative stress and glucose deprivation (Ferreira and Caceres, 1991;Lorenzo et al, 1992;Behl et al, 1997;Goodman et al, 1996;Green et al, 2000;Zhang et al, 2001). More recently, estradiol has also been shown to promote neurogenesis within the adult hippocampal formation (Gould et al, 2000).…”

Involvement of Insulin/Phosphoinositide 3-Kinase/Akt Signal Pathway in 17β-Estradiol-mediated Neuroprotection

“…For many years, it has been known that ␤E2 promotes viability and survival of neurons in primary neuronal cultures. Addition of ␤E2 to defined culture media increased the viability, survival, and differentiation of primary neuronal cultures from different brain areas including amygdala (29), hypothalamus (30), and neocortex (31). However, the role of estrogen in the protection of retinal neurons is not well understood.…”

Estrogens Cross-Talk to α1b-Adrenergic Receptors

“…We performed many of the present experiments using a neural cell line because it is well known that estrogens have neuroprotective properties (Lorenzo et al, 1992;Yu et al, 2004). Addition of 17␤-estradiol increased the viability, survival, and differentiation of primary neuronal cultures.…”