Regulation of Dendritic Spine Density in Cultured Rat Hippocampal Neurons by Steroid Hormones

Abstract

The effects of gonadal steroid hormones on dendritic spines were studied in hippocampal neurons that were dissociated and grown in culture for 2-3 weeks. Exposure to estradiol caused up to a twofold increase in dendritic spine density in these neurons. The effect of estradiol was stereospecific and blocked by the steroid antagonist tamoxifen. The estradiolinduced rise in spine density was blocked by the NMDA antagonist APV, but not by the AMPA/KA antagonist DNQX. The estradiol-induced rise in spine density was blocked by the serine/threonine kinase inhibitor H7, but not by the tyrosine kinase inhibitor genestein, and was partially mimicked by PMA, an activator of protein kinase C. Estradiol also caused an increase in the fluorescence intensity of synaptophysinimmunoreactive terminals, corresponding to presynaptic boutons. Finally, estradiol caused a rise in [Ca] i reactivity of the cultured neurons to topical application of glutamate. These studies are the first to examine receptor and second messenger regulation of dendritic spines, and they illustrate the viability of cultured neurons as a powerful test system to address issues related to the regulation of dendritic spine maturation.Key words: dendritic spines; estradiol; culture; hippocampal neurons; calcium; plasticity Dendritic spines are the loci of synaptic interactions among central neurons. Their susceptibility to changes in afferent stimulation makes dendritic spines the prime candidates for serving the longterm morphological substrates of neuronal plasticity. In the hippocampus, which has been associated with learning and memory, spines may be essential for the induction, associativity, specificity, and endurance of long-term potentiation (Harris and Kater, 1994). Despite extensive interest in dendritic spines, progress toward understanding the mechanisms governing their generation and morphological plasticity has been rather slow. This is attributable partly to their minute size, which is at the limit of optical resolution, and their large density and heterogeneity in neurons of the intact brain.Dissociated cultures of hippocampal neurons offer a viable and convenient means for studying the factors that regulate the development and functions of dendritic spines. The two-dimensional nature of cultured neurons, as well as the relative sparseness of dendritic spines in culture, which are approximately one third to one fourth the density of those neurons in vivo, makes their study technically achievable (Papa et al., 1995; Segal, 1995a,b).It has been observed recently that the density of dendritic spines in the intact rat hippocampus undergoes marked variations during the estrous cycle. A higher density of dendritic spines coincides with high levels of estrogen , and the addition of estradiol to ovariectomized rats causes a marked increase in spine density (Woolley and McEwen, 1994). Circulating estrogens are prevalent during neonatal development and so may be active in neonatal spine formation. In fact, estrogens may be regulating synaptic plasticity i...

Estrogen-like Activity of Tamoxifen and Raloxifene on NMDA Receptor Binding and Expression of its Subunits in Rat Brain

“…In contrast, CI-628 has an estrogen-like agonist activity on brain monoamine oxidase in corticomedial amygdala (reduction of activity) and on choline acetylase in the preoptic area (increase of activity) (Luine and McEwen 1977). Moreover, tamoxifen was also shown to block the effect of estradiol on dendritic spines of hippocampal neurons (Murphy and Segal 1996). The raloxifene analogue LY117018 has an estrogen-like action on neuroendocrine opiatergic pathways when administered alone in ovariectomized rats, whereas it exerts an anti-estrogenic effect in fertile or ovariectomized rats treated with 17␤-estradiol (Genazzani et al 1999).…”

Environmental enrichment reduces the mnemonic and neural benefits of estrogen

“…For example, increases in synaptophysin, a presynaptic protein located in the membranes of neurotransmitter-containing vesicles (Calhoun et al, 1996;Schlaf et al, 1996) have been noted following estrogen treatment. Estradiol increases synaptophysin expression in cultured hippocampal neurons and elevates synaptophysin immunoreactivity in the cornu ammonis (CA) 1 region of the rat hippocampus (Brake et al, 2001;Murphy and Segal, 1996;PozzoMiller et al, 1999b). Furthermore, estrogen increases levels of synaptophysin in aged female mice, a finding which has been associated with enhanced hippocampal-dependent learning (Frick et al, 2002).…”

Ovariectomy up-regulates neuronal neurofilament light chain mRNA expression with regional and temporal specificity

“…These include alterations in a variety of intracytoplasmic organelles such as the endoplasmic reticulum, mitochondria and lysosomes, as well as depletion of microtubules and ®laments in dendritic processes. 7 In addition, GS (particularly estrogen) concentration reduction provoke dendritic spines withdrawal in both hippocampal 16,40 and septal 38 neuronal cultures. NF-L mRNA levels are reduced in early axonal degeneration, 28 in aging, 46 as well as in several neurodegenerative diseases.…”

“…12 The above considerations highlight the need to examine the longer term consequences of OVx, particularly as they impact upon the cytoskeleton, since this could produce alterations in neuronal architecture, possibly even resulting in lost neuronal connectivity and in the formation of pathological structures. Estrogens can modulate the cytoskeletal architecture of susceptible neurons, since both dendritic spine density in hippocampal pyramidal neurons in rodents, 16,40 as well as outgrowth of neurites in cell cultures 5,6,31 have been shown to increase as a function of higher estrogen levels.AD, the most common neurodegenerative cause of dementia in old age, is characterized primarily by a degeneration of cholinergic neurons in the basal forebrain. As a result, a cholinergic de®cit develops in relevant projection areas, particularly the neocortex and the hippocampus, with resultant cognitive de®cits involving Ovariectomy up-regulates neuronal NF-L mRNA 629 629…”