Stephen E. Alves

Ovarian steroids and the brain Implications for cognition and aging

Article abstract-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 may help to 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. NEUROLOGY 1997;48(Suppl 7): S8-S15

Ultrastructural localization of estrogen receptor β immunoreactivity in the rat hippocampal formation

Several lines of evidence indicate that estrogen affects hippocampal synaptic plasticity through rapid nongenomic mechanisms, possibly by binding to plasma membrane estrogen receptors (ERs). We have previously shown that ERα immunoreactivity (ir) is in select interneuron nuclei and in several extranuclear locations, including dendritic spines and axon terminals, within the rat hippocampal formation (Milner et al., [ 2001 ] J Comp Neurol 429:355). The present study sought to determine the cellular and subcellular locations of ERβ‐ir. Coronal hippocampal sections from diestrus rats were immunolabeled with antibodies to ERβ and examined by light and electron microscopy. By light microscopy, ERβ‐ir was primarily in the perikarya and proximal dendrites of pyramidal and granule cells. ERβ‐ir was also in a few nonprincipal cells and scattered nuclei in the ventral subiculum and CA3 region. Ultrastructural analysis revealed ERβ‐ir at several extranuclear sites in all hippocampal subregions. ERβ‐ir was affiliated with cytoplasmic organelles, especially endomembranes and mitochondria, and with plasma membranes primarily of principal cell perikarya and proximal dendrites. ERβ‐ir was in dendritic spines, many arising from pyramidal and granule cell dendrites. In both dendritic shafts and spines, ERβ‐ir was near the perisynaptic zone adjacent to synapses formed by unlabeled terminals. ERβ‐ir was in preterminal axons and axon terminals, associated with clusters of small, synaptic vesicles. ERβ‐labeled terminals formed both asymmetric and symmetric synapses with dendrites. ERβ‐ir also was detected in glial profiles. The cellular and subcellular localization of ERβ‐ir was generally similar to that of ERα, except that ERβ was more extensively found at extranuclear sites. These results suggest that ERβ may serve primarily as a nongenomic transducer of estrogen actions in the hippocampal formation. J. Comp. Neurol. 491:81–95, 2005.

Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Ultrastructural evidence that androgen receptors are located at extranuclear sites in the rat hippocampal formation

Like estrogens in female rats, androgens can affect dendritic spine density in the CA1 subfield of the male rat hippocampus [J Neurosci 23:1588 (2003)]. Previous light microscopic studies have shown that androgen receptors (ARs) are present in the nuclei of CA1 pyramidal cells. However, androgens may also exert their effects through rapid non-genomic mechanisms, possibly by binding to membranes. Thus, to investigate whether ARs are at potential extranuclear sites of ARs, antibodies to ARs were localized by light and electron microscopy in the male rat hippocampal formation. By light microscopy, AR immunoreactivity (-ir) was found in CA1 pyramidal cell nuclei and in disperse, punctate processes that were most dense in the pyramidal cell layer. Additionally, diffuse AR-ir was found in the mossy fiber pathway. Ultrastructural analysis revealed AR-ir at several extranuclear sites in all hippocampal subregions. AR-ir was found in dendritic spines, many arising from pyramidal and granule cell dendrites. AR-ir was associated with clusters of small, synaptic vesicles within preterminal axons and axon terminals. Labeled preterminal axons were most prominent in stratum lucidum of the CA3 region. AR-containing terminals formed asymmetric synapses or did not form synaptic junctions in the plane of section analyzed. AR-ir also was detected in astrocytic profiles, many of which apposed terminals synapsing on unlabeled dendritic spines or formed gap junctions with other AR-labeled or unlabeled astrocytes. Collectively, these results suggest that ARs may serve as both a genomic and non-genomic transducer of androgen action in the hippocampal formation.

Immunocytochemical localization of nuclear estrogen receptors and progestin receptors within the rat dorsal raphe nucleus

Estradiol and progesterone modulate central serotonergic activity; however, the mechanism(s) of action remain unclear. Recently, estradiol‐induced progestin receptors (PRs) have been localized within the majority of serotonin (5‐HT) neurons in the female macaque dorsal raphe nucleus (DRN; Bethea [1994] Neuroendocrinology 60:50–61). In the present study, we investigated whether estrogen receptors (ERs) and/or PRs exist within 5‐HT and/or non‐5‐HT cells in the female and male rat DRN and whether estradiol treatment alters the expression of these receptors. Young adult female and male Sprague‐Dawley rats were gonadectomized, and 1 week later, half of the animals received a subcutaneous Silastic implant of estradiol‐17β. Animals were transcardially perfused 2 days later with acrolein and paraformaldehyde, and sequential dual‐label immunocytochemistry was performed on adjacent sections by using either a PR antibody or an ERα antibody. This was followed by an antibody to either the 5‐HT‐synthesizing enzyme, tryptophan hydroxylase (TPH), or to the astrocytic marker, glial fibrillary acidic protein (GFAP). Cells containing immunoreactivity (ir) for nuclear ERs or PRs were identified within the rat DRN in a region‐specific distribution in both sexes. No colocalization of nuclear ER‐ir or PR‐ir with cytoplasmic TPH‐ir or GFAP‐ir was observed in either sex or treatment, indicating that the steroid target cells are neither 5‐HT neurons nor astrocytes. Females were found to have approximately 30% more PR‐labeled cells compared with males throughout the DRN (P < 0.05), but no sex difference was detected in the number of neurons demonstrating ER‐ir. In both sexes, 2 days of estradiol exposure decreased the number of cells with ER‐ir, whereas it greatly increased the number of cells containing PR‐ir in several DRN regions (P < 0.005). Collectively, these findings demonstrate the existence of nonserotonergic cells that contain nuclear ERs or PRs within the female and male rat DRN, including estradiol‐inducible PRs. These findings point to a species difference in ovarian steroid regulation of 5‐HT activity between the macaque and the rat, perhaps transsynaptically via local neurons in the rat brain. J. Comp. Neurol. 391:322–334, 1998.

Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Subcellular relationships between cholinergic terminals and estrogen receptor-α in the dorsal hippocampus

Cholinergic septohippocampal neurons are affected by circulating estrogens. Previously, we found that extranuclear estrogen receptor‐α (ERα) immunoreactivity in presynaptic profiles had an overlapping distribution with cholinergic afferents in the rat hippocampal formation. To determine the subcellular relationships between cholinergic presynaptic profiles and ERα, hippocampal sections were dually immunolabeled for vesicular acetylcholine transporter (VAChT) and ERα and examined by electron microscopy. Within the hippocampal formation, immunoreactivities for VAChT and ERα both were presynaptic, although their subcellular targeting was distinct. VAChT immunoreactivity was found exclusively within presynaptic profiles and was associated with small synaptic vesicles, which usually filled axon terminals. VAChT‐labeled presynaptic profiles were most concentrated in stratum oriens of the hippocampal CA1 region and dentate inner molecular layer and hilus. In contrast, ERα immunoreactivity was found in clusters affiliated either with select vesicles or with the plasmalemma within preterminal axons and axon terminals. ERα‐immunoreactive (IR) presynaptic profiles were more evenly distributed between hippocampal lamina than VAChT‐IR profiles. Quantitative ultrastructural analysis revealed that VAChT‐IR presynaptic profiles contained ERα immunoreactivity (ranging from 3% to 17%, depending on the lamina). Additionally, VAChT‐IR presynaptic profiles apposed ERα‐IR dendritic spines, presynaptic profiles, and glial profiles; many of the latter two types of profiles abutted unlabeled dendritic spines that received asymmetric (excitatory‐type) synapses from unlabeled terminals. The presence of ERα immunoreactivity in cholinergic terminals suggests that estrogen could rapidly and directly affect the local release and/or uptake of acetylcholine. The affiliation of cholinergic terminals with excitatory terminals near ERα‐labeled dendritic spines or glial profiles suggests that alterations in acetylcholine release could indirectly affect estrogen‐modulated structural plasticity. J. Comp. Neurol. 463:390–401, 2003.

Estrogen-regulated progestin receptors are found in the midbrain raphe but not hippocampus of estrogen receptor alpha (ER?) gene-disrupted mice

Estrogen and progesterone may modulate serotonergic function through intracellular receptors, alpha (ERα) and/or beta (ERβ), and the progestin receptor (PR). Studies in macaque and rat suggest species differences in steroid action. Presently, we examined the mouse. To identify whether ERα is involved in estrogen induction of PR in midbrain raphe, we studied the ERα gene‐disrupted (αERKO) mouse. The hippocampus was examined as another estrogen/progestin‐sensitive brain area reported to express ERα, ERβ, and PR. Female and male homozygous αERKO and wildtype mice were gonadectomized and given estradiol benzoate or vehicle. Dual‐label immunocytochemistry was performed for PR or ERα and the serotonin‐synthesizing enzyme, tryptophan hydroxylase (TPH). Cells exhibiting PR immunoreactivity (PR‐ir) or ERα‐ir were observed in dorsal and median raphe and hippocampus in both sexes. No ERα‐ir cells were observed in αERKO brains. In raphe, PR‐ir or ERα‐ir often colocalized with TPH‐ir. Thus, estrogen and progesterone may directly modulate gene expression in select serotonergic neurons via ERα and PR in female and male mice. Estrogen significantly increased the number of PR‐ir cells, and the percentage of PR‐ir cells colocalizing TPH‐ir in both raphe nuclei, regardless of sex and genotype. Although less among αERKO mice, the significant estrogen induction of PRs implicates the involvement of another ER, perhaps ERβ. In hippocampus, distinct estrogen‐induced PR‐ir cells were observed only in wildtype animals, demonstrating an ERα‐mediated event in this forebrain region. Collectively, these findings suggest that estrogen can regulate the expression of one gene (the PR) via multiple mechanisms, based upon brain region. J. Comp. Neurol. 427:185–195, 2000.

Estradiol and ERβ agonists enhance recognition memory, and DPN, an ERβ agonist, alters brain monoamines

Effects of estradiol benzoate (EB), ERα-selective agonist, propyl pyrazole triol (PPT) and ERβ-selective agonists, diarylpropionitrile (DPN) and Compound 19 (C-19) on memory were investigated in OVX rats using object recognition (OR) and placement (OP) memory tasks. Treatments were acute (behavior 4h later) or sub chronic (daily injections for 2 days with behavior 48 h later). Objects were explored in sample trials (T1), and discrimination between sample (old) and new object/location in recognition trials (T2) was examined after 2-4h inter-trial delays. Subjects treated sub chronically with EB, DPN, and C-19, but not PPT, discriminated between old and new objects and objects in old and new locations, suggesting that, at these doses and duration of treatments, estrogenic interactions with ERβ contribute to enhancements in recognition memory. Acute injections of DPN, but not PPT, immediately after T1, also enhanced discrimination for both tasks (C19 was not investigated). Effects of EB, DPN and PPT on anxiety and locomotion, measured on elevated plus maze and open field, did not appear to account for the mnemonic enhancements. Monoamines and metabolites were measured following DPN treatment in subjects that did not receive behavioral testing. DPN was associated with alterations in monoamines in several brain areas: indexed by the metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), or the MHPG/norepinephrine (NE) ratio, NE activity was increased by 60-130% in the prefrontal cortex (PFC) and ventral hippocampus, and NE activity was decreased by 40-80% in the v. diagonal bands and CA1. Levels of the dopamine (DA) metabolite, homovanillic acid (HVA), increased 100% in the PFC and decreased by 50% in the dentate gyrus following DPN treatment. The metabolite of serotonin, 5-hydroxyindole acetic acid (5-HIAA), was increased in the PFC and CA3, by approximately 20%. No monoaminergic changes were noted in striatum or medial septum. Results suggest that ERβ mediates sub chronic and acute effects of estrogens on recognition memory and that memory enhancements by DPN may occur, in part, through alterations in monoaminergic containing systems primarily in PFC and hippocampus.

Estrogen receptor-β regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe

BACKGROUND Distinct expression patterns of estrogen receptor (ER)-alpha and ER-beta are displayed in the murine central nervous system. ER-beta is the predominant form of the receptor expressed in the murine midbrain dorsal raphe nucleus (DRN). Tryptophan hydroxylase (TPH) is abundantly expressed in the serotonergic neurons of the DRN and is regulated by estrogen in both the monkey and the guinea pig. METHODS In this study we used immunocytochemistry to show that ER-beta and TPH are colocalized in the serotonergic cells of the murine DRN. We utilized the ER-alpha and ER-beta gene deletion mouse models and in situ hybridization to demonstrate that ER-beta is responsible for regulating TPH1 mRNA expression. RESULTS Estrogen increased TPH1 mRNA expression in the DRN of wild type and ER-alpha knockout mice (alpha-ERKO) but not ER-beta knockouts (beta-ERKO). CONCLUSIONS These data indicate that ER-beta is responsible for mediating estrogen regulated TPH1 expression in the murine DRN.