Previous studies have shown that estradiol induces new dendritic spines and synapses on hippocampal CA1 pyramidal cells. We have assessed the consequences of estradiolinduced dendritic spines on CA1 pyramidal cell intrinsic and synaptic electrophysiological properties. Hippocampal slices were prepared from ovariectomized rats treated with either estradiol or oil vehicle. CA1 pyramidal cells were recorded and injected with biocytin to visualize spines. The association of dendritic spine density and electrophysiological parameters for each cell was then tested using linear regression analysis. We found a negative relationship between spine density and input resistance; however, no other intrinsic property measured was significantly associated with dendritic spine density. Glutamate receptor autoradiography demonstrated an estradiol-induced increase in binding to NMDA, but not AMPA, receptors. We then used input/output (I/O) curves (EPSP slope vs stimulus intensity) to determine whether the sensitivity of CA1 pyramidal cells to synaptic input is correlated with dendritic spine density. Consistent with the lack of an estradiol effect on AMPA receptor binding, we observed no relationship between the slope of an I/O curve generated under standard recording conditions, in which the AMPA receptor dominates the EPSP, and spine density. However, recording the pharmacologically isolated NMDA receptor-mediated component of the EPSP revealed a significant correlation between I/O slope and spine density. These results indicate that, in parallel with estradiol-induced increases in spine/synapse density and NMDA receptor binding, estradiol treatment increases sensitivity of CA1 pyramidal cells to NMDA receptor-mediated synaptic input; further, sensitivity to NMDA receptor-mediated synaptic input is well correlated with dendritic spine density. Key words: estradiol; dendritic spines; hippocampal slice; CA1 pyramidal cells; biocytin; autoradiography; NMDA receptorSince the initial anatomical description of dendritic spines by Ramon y Cajal in the late 1800s, many investigators have speculated about dendritic spine function. Spines have been proposed to play primarily connective, electrical, and /or biochemical roles in neuronal physiology (for review, see Koch and Zador, 1992;Horner, 1993;Harris and Kater, 1994). Recent efforts to understand dendritic spine function based on imaging of dye-labeled spiny dendrites have yielded valuable information, particularly with regard to spines' potential for Ca 2ϩ compartmentalization (Guthrie et al., 1991;Muller et al., 1991). Such findings have been incorporated into proposals for dendritic spine function in synaptic integration (see, for example, Yuste and Denk, 1995) and neuroprotection (Harris and Kater, 1994;Segal, 1995). An additional approach to exploring dendritic spine function, which has not been used to date, is to assess the functional consequences of adding dendritic spines to the dendrites of spiny neurons.Hippocampal CA1 pyramidal cells are an ideal population of neurons in w...

Estradiol treatment increases the number of NMDA receptor binding sites, and changes evoked synaptic currents in a manner consistent with a steroid-induced functional enhancement of NMDA receptors in rat hippocampus. In this study, we investigate the cellular mechanisms of estradiol-induced NMDA receptor regulation at the protein and mRNA levels in ovariectomized rats treated with ovarian steroids using immunocytochemical and in situ hybridization techniques. Confocal laser scanning microscopy was used to quantify alterations in immunofluorescence intensity levels of NMDAR1 subunit proteins within neuronal somata and dendrites of discrete hippocampal fields, whereas in parallel, in situ hybridization was used to examine NMDAR1 mRNA levels in corresponding hippocampal regions. The data indicate that estradiol treatment in ovariectomized rats significantly increases immunofluorescence intensity levels in comparison with nonsteroid treated ovariectomized rats within the somata and dendrites of CA1 pyramidal cells and, to a lesser extent, within the granule cell somata of the dentate gyrus. In contrast, such alterations in immunofluorescence intensity occur without concomitant changes in mRNA hybridization levels. Thus, these data suggest that estradiol modulates NMDA receptor function via post-transcriptional regulation of the NMDAR1 subunit protein. The increase in immunofluorescence intensity may reflect an increase in the concentration of the subunit protein, which could account for estrogen-induced changes in pharmacological and physiological properties of the NMDA receptor.

Ovarian steroids have many effects on the brain throughout the lifespan, beginning during gestation and continuing into senescence. These hormones affect areas of the brain that are not primarily involved in reproduction, such as the basal forebrain, hippocampus, caudate putamen, midbrain raphe, and brainstem locus coeruleus. Here we discuss three effects of estrogens and progestins that are especially relevant to memory processes and identify hormonal alterations associated with aging and neurodegenerative diseases. First, estrogens and progestins regulate synaptogenesis in the CA1 region of the hippocampus during the 4- to 5-day estrous cycle of the female rat. Formation of new excitatory synapses is induced by estradiol and involves N-methyl-D-aspartate (NMDA) receptors, whereas synaptic downregulation involves intracellular progestin receptors. Second, there are developmentally programmed sex differences in the hippocampal structure that mat help explain why male and female rats use different strategies to solve spatial navigation problems. During the period of development when testosterone is elevated in the male, aromatase and estrogen receptors are transiently expressed in the hippocampus. Recent data on behavior and synapse induction strongly suggest that this pathway is involved in the masculinization or defeminization of hippocampal structure and function. Third, ovarian steroids have effects throughout the brain, including effects on brainstem and midbrain catecholaminergic neurons, midbrain serotonergic pathways, and the basal forebrain cholinergic system. Regulation of the serotonergic system appears to be linked to the presence of estrogen- and progestin-sensitive neurons in the midbrain raphe, whereas the ovarian steroid influence on cholinergic function involves induction of choline acetyltransferase and acetylcholinesterase according to a sexually dimorphic pattern. Because of these widespread influences on these various neuronal systems, it is not surprising that ovarian steroids produce measurable cognitive effects after ovariectomy and during aging.

Evidence exists for the localization of the newly identified estrogen receptor ␤ (ER␤) within the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), regions which lack ER␣. Presently, we investigate whether ER␤-like-immunoreactivity (-ir) is found within cells of several major neuropeptide systems of these regions. Young adult Sprague-Dawley rats were ovariectomized (OVX), and 1 week later half of the animals received estradiol-17␤ (E). Dual-label immunocytochemistry was performed on adjacent sections by using an ER␤ antibody, followed by an antibody to either oxytocin (OT), arginine-vasopressin (AVP), or corticotropin releasing hormone. Nuclear ER␤-ir was identified within SON and retrochiasmatic SON, and in specific PVN subnuclei: medial parvicellular part, ventral and dorsal zones, dorsal and lateral parvicellular parts, and in the posterior magnocellular part, medial and lateral zones. However, the ER␤-ir within magnocellular areas was noticeably less intense. OT-͞ER␤-ir colocalization was confirmed in neurons of the parvicellular subnuclei, in both OVX and OVX؉E brains (Ϸ50% of OT and 25% of ER␤-labeled cells between bregma ؊1.78 and ؊2.00). In contrast, few PVN parvicellular neurons contained both AVP-and ER␤-ir. As well, very little overlap was observed in the distribution of cells containing corticotropin releasing hormone-or ER␤-ir. In the SON, most nuclear ER␤-ir colocalized with AVP-ir, whereas few OT-͞ ER␤-ir dual-labeled cells were observed. These findings suggest that estrogen can directly modulate specific OT and AVP systems through an ER␤-mediated mechanism, in a tissuespecific manner.The newly identified estrogen receptor ␤ (ER␤) has been shown to exist in rat (1), human (2), and mouse (3), but the physiological role(s) of this receptor remain(s) unknown. The ligand binding characteristics have been found to be generally similar to those of the ''original'' ER, now known as ER␣, despite only a 55-60% homology in the C-terminal ligand binding domain of the two receptors (1-4). Likewise, ER␤ appears capable of activating the expression of an estrogen response element-containing reporter gene construct, in a hormone-dependent manner, at very low hormone concentrations (1-3). This finding is not surprising, considering that the DNA binding domains of the two isoforms are 95-97% homologous. However, the remaining domains of these two steroid receptors show no homology, and recent evidence suggests that ER␣ and ER␤ possess distinct transactivation functions (3, 5).Another clue for a distinct role for this ''second'' ER may be its anatomical distribution. Although there appears to be some overlap, the distribution of the two receptor subtypes appear to be quite different, based upon recent descriptions of ER␤ transcript localization in the rat (4). In the rat brain specifically, the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) have been identified as having large concentrations of cells containing ER␤ mRNA (6, 7) or immunoreactivity (8). Although [ 3 H]estrogen-co...

Estrogen regulates the synaptic plasticity and physiology of the hippocampus as well as learning behaviors that are mediated by the hippocampus. The density of dendritic spines and synapses, the number of N-methyl-D-aspartate (NMDA) binding sites, the levels of NMDA receptor subunit NR1 protein, muscimol binding to the gamma-amino butyric acid (GABA)A receptor, and levels of glutamic acid decarboxylase message in the CA1 region of the hippocampus are altered with estrogen treatment. In addition, some of these parameters exhibit sex differences in their response to estrogen treatment. To establish that estrogen can have a direct effect on the hippocampus and to determine whether or not sex differences in estrogen responsiveness are due to sex differences in estrogen receptor (ER) levels, we used immunocytochemistry with the AS409 antibody to map the location of ER-immunoreactive (ER-ir) cells in the hippocampus of male and female rats. We found that (1) the ERs appear to be in interneurons rather than pyramidal or granule cell neurons, (2) ER-ir cells are located in greatest concentration in the hilus of the dentate gyrus and the stratum radiatum of the CA1 region, (3) the density of ER-ir cells exhibits a rostral to caudal gradient in the hilus and the CA1 regions, (4) there are no sex differences in either the number or immunostaining intensity of ER-ir cells in the hippocampus, (5) the ER levels are down-regulated by estrogen in both male and female rats, and (6) the mean intensity of staining for the ER-ir cells in the hippocampus is about 25% of that in the ER-ir cells of the hypothalamus. From this, we can conclude that estrogen can have a direct effect on hippocampal neurons and that any sex differences in estrogen responsiveness is due to something other than sex differences in ER levels or function in the hippocampus.