Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons. P revious studies have shown that estrogen increases the density of synaptic input to hippocampal CA1 pyramidal cells in adult female rats. The density of both dendritic spines (e.g., refs. 1 and 2) and spine synapses (3) on CA1 pyramidal cells is greater in ovariectomized estrogen-treated rats than in ovariectomized controls. Consistent with the fact that dendritic spines are sites of excitatory input, electrophysiological studies show that the estrogen-induced increase in spine density is paralleled by enhanced sensitivity of CA1 pyramidal cells to excitatory synaptic input (4).Based on a serial electron-microscopic study of presynaptic boutons and postsynaptic dendritic spines, Woolley et al. (5) proposed that new spines induced by estrogen form synapses primarily with preexisting presynaptic boutons. This suggestion was based on the following argument. Presynaptic boutons afferent to CA1 dendritic spines can be divided into 2 classes (6): single synapse boutons (SSBs), which are synaptically connected to one dendritic spine and multiple synapse boutons (MSBs), which are synaptically connected to more than one spine. Analysis of CA1 presynaptic boutons showed an estrogeninduced increase in the relative frequency of MSBs to SSBs as well as an increase in the average number of synapses each MSB forms (5). Using these data, Woolley and colleagues calculated that for any given number of presynaptic boutons, there were 25% more synapses formed on CA1 spines in estrogen-treated animals. This value is very similar to the percentage incre...

Although the classical mechanism of estrogen action involves activation of nuclear transcription factor receptors, estrogen also has acute effects on neuronal signaling that occur too rapidly to involve gene expression. These rapid effects are likely to be mediated by extranuclear estrogen receptors associated with the plasma membrane and/or cytoplasmic organelles. Here we used a combination of serialsection electron microscopic immunocytochemistry, immunofluorescence, and Western blotting to show that estrogen receptor-␣ is associated with clusters of vesicles in perisomatic inhibitory boutons in hippocampal CA1 and that estrogen treatment mobilizes these vesicle clusters toward synapses. Estrogen receptor-␣ is present in approximately one-third of perisomatic inhibitory boutons, and specifically in those that express cholecystokinin, not parvalbumin. We also found a high degree of extranuclear estrogen receptor-␣ colocalization with neuropeptide Y. Our results suggest a novel mode of estrogen action in which a subset of vesicles within a specific population of inhibitory boutons responds directly to estrogen by moving toward synapses. The mobilization of these vesicles may influence acute effects of estrogen mediated by estrogen receptor-␣ signaling at inhibitory synapses.

Despite the many effects of estrogen in the hippocampus, there has been little evidence that hippocampal principal cells express nuclear estrogen receptors. In the hippocampus, the alpha form of the nuclear estrogen receptor (ER alpha) has been localized to sparsely distributed cells with the morphological characteristics of inhibitory interneurons. Because inhibitory neurons may be involved in the effects of estrogen on hippocampal principal cells, quantitative description of ER alpha expression in gamma-aminobutyric acid (GABA)ergic (inhibitory) and non-GABAergic cells of the hippocampus is a key step in understanding the mechanism(s) of estrogen action on hippocampal circuitry. We used single and double-label immunohistochemistry for ER alpha and glutamic acid decarboxylase (GAD; a marker of GABAergic neurons) to determine the numbers and distributions of hippocampal GABAergic and non-GABAergic neurons that express ER alpha in the adult female rat. We found many more ER alpha-expressing cells in the hippocampus than any previous study and observed distinct dorsal vs. ventral differences in hippocampal ER alpha expression. In the dorsal hippocampus, most ER alpha-positive cells were also GAD positive; however, ER alpha was expressed in only a subset of GAD-positive cells. Double-labeled cells were concentrated at the border between str. radiatum and str. lacunosum-moleculare. In the ventral hippocampus, we found a very high number of ER alpha-positive cells, the majority of which were not immunoreactive for GAD and are likely to be pyramidal cells. These findings suggest that ER alpha can mediate the effects of estrogen primarily in GABAergic neurons in the dorsal hippocampus and in both GABAergic and non-GABAergic neurons in the ventral hippocampus.

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This study examined the central effects of rat prepro-TRH 178-199, a peptide with corticotropin release inhibiting activity at the pituitary, on the Porsolt forced swim test (FST) of depressive behavior in rats. Subacute intracerebroventricular administration of prepro-TRH 178-199 dose-responsively reduced floating and increased active behaviors in the FST. Chronic administration of 6 microg/kg prepro-TRH 178-199 decreased floating more than subacute treatment, but there were no significant differences between chronic and subacute treatment effects on active behavior. Biological activity of this peptide resides in the C-terminal fragment as prepro-TRH 178-199 and prepro-TRH 191-199 were equally potent in the FST. These data suggest that endogenous prepro-TRH 178-199 with its antidepressant-like activity might contribute to the etiology or manifestation of depressive behavior.