Marilyn M. Miller

Estrogen effects on object memory and cholinergic receptors in young and old female mice

We investigated whether object recognition memory is modulated by estrogen in young (5 month) and aged (24 month) female C57Bl/6J mice, and if cholinergic muscarinic receptors might contribute to this response. Mice that were ovariectomized, or ovariectomized plus estradiol-treated three weeks before behavioral testing or quantitative autoradiography were compared to intact mice. Memory for a previously encountered object deteriorated significantly between 3 and 6h after initial exposure, regardless of animal age. In both young and aged mice, estradiol-treated mice showed significantly greater recall than did ovariectomized mice. In both age groups, the apparent number of [(3)H]pirenzepine/M(1)-like and [(3)H]AFDX384/M(2)-like muscarinic receptor binding sites was reduced in the basal forebrain as well as its projection areas following ovariectomy, but this decrease was not alleviated by estrogen. Aging poorly affected object memory, but reduced muscarinic binding in some cortical subregions and in the caudate nucleus. These findings suggest that estrogen effects on memory in C57Bl/6J mice are not due to changes in the number of muscarinic receptors.

Estrogens (E2) regulate expression and response of 1,25-dihydroxyvitamin D3 receptors in bone cells: changes with aging and hormone deprivation ☆

Studies on the effect of estrogens (E(2)) on the expression of vitamin D receptor (VDR) and its bioresponse in bone have demonstrated that E(2) modulate activity and increase the number of VDRs in vitro; however, no in vivo studies have been pursued to assess this interaction. Our study identifies the changes in the number of VDR-expressing cells in bone of C57BL/6J young and old oophorectomized mice (4 and 24 months) with and without 17beta estradiol (E(2)) replacement. A total of 36 mice were sacrificed; both tibiae and femora were isolated and VDR expression was quantified by Northern blot, immunohistochemistry, immunofluorescence, and flow cytometry. Among the intact mice there was a significant difference in the number of VDR-expressing osteoblasts between young (68%) and old (56%) (p<0.04). In young oophorectomized mice the number of VDR-expressing osteoblasts decreased from 68% to 46% after oophorectomy and recovered to 72% after E(2) administration (p<0.02), while in the group of old mice, the number of VDR-expressing osteoblasts decreased from 56% to 48% after oophorectomy (p<0.01) and recovered to 85% after E(2) administration (p<0.001). Our results show that VDR expression in bone decreases with aging and estrogen deprivation but recovers after E(2) supplementation in both young and old mice with a more significant level of response in older bone. To evaluate the level of VDR bioresponse to E(2) we assessed the effect of E(2) supplementation to human osteoblasts (N-976) in vitro. Northern blot showed a significant up-regulation of VDR expression in E(2) treated cells as compared to non-treated cells (p<0.05). We also assessed the previously known anti-apoptotic effect of vitamin D in osteoblasts in vitro after serum deprivation by using either E(2), E(2)+1,25(OH)(2)D(3), or 1,25(OH)(2)D(3) alone. We found a lower number of apoptotic cells and longer cell survival after 48 h of treatment with 1,25(OH)(2)D(3)+E(2) as compared to 1,25(OH)(2)D(3) or E(2) alone (p<0.002). In summary, our results demonstrate that E(2) increases VDR expression in bone in vivo and potentiate the bioresponse of VDR in osteoblasts in vitro.

THEORETICAL BASIS FOR THE BENEFIT OF POSTMENOPAUSAL ESTROGEN SUBSTITUTION

Women are being presented with an increasing number of choices for health care management as they move through the aging process. Estrogen has positive effects on mood, sexual function, target end organs and cognitive function, and may play an important role in the etiology of Alzheimers Disease by acting to prevent amyloid plaque formation, oxidative stress, or deterioration of the cholinergic neurotransmitter system. The benefits of estrogen therapy for osteoporosis, the cardiovascular system, and lipid metabolism are far reaching, but the possibility of developing breast cancer later in life is also relevant. Understanding the mechanisms for the action of the estrogens, anti-estrogens, and the selective estrogen receptor modulators, and possible alternative routes of symptom management for some menopausal events is important to make appropriate decisions on choice of therapy. This review discusses the theoretical basis for estrogens actions in the management of the postmenopausal stage of the life cycle.

Loss of LH-RH neurons in the rostral forebrain of old female C57BL/6J mice

This study reports the quantitative immunohistochemical distribution of luteinizing hormone-releasing hormone (LH-RH) neurons within the rostral forebrain of young (5-6 month) and old (26-28 month) C57BL/6J mouse. Old mice demonstrated a significant (18%) loss of LH-RH-containing neurons (p less than 0.001, ANOVA). The most striking losses involved the preoptic area (24%) and more rostral regions (26%). The presence of pituitary or mesenteric tumors in older mice did not affect the density of LH-RH neurons. These observations indicate that LH-RH neurons comprise part of the neuronal population previously reported to be reduced in the preoptic area of older mice (4).

Effects of Ovariectomy and Estradiol Replacement on the Binding of 125I-Neurotensin in Rat Suprachiasmatic Nucleus

Distribution and density of specific high-affinity 125I-neurotensin-binding sites were examined by light microscopic radioautography in the suprachiasmatic nucleus (SCN) of normal cycling, ovariectomized, or ovariectomized and estradiol-implanted female rats. In all three experimental groups, intense 125I-neurotensin labeling was detected within the ventrolateral component of the SCN. Whereas the topographic distribution and spread of the label was similar between each group, the density of the label was significantly higher (mean increase: 122%) in ovariectomized than in normally cycling females within the rostral third of the SCN. This effect was no longer apparent in females chronically implanted with estradiol at the time of gonadectomy. These results indicate that plasma gonadal steroids may regulate neurotensin receptors in the rat SCN. It is suggested that this mechanism might be implicated in the feedback control of gonadotropin and prolactin secretion.

Synaptic ribbon populations in the pineal gland of the rhesus monkey (Macaca mulatta).

SummaryPinealocytes of rhesus monkeys that had been ovariectomized and given intramuscular injections of 250 μg estradiol-benzoate for 3 consecutive days tended to have more synaptic ribbons (SR) and exhibited a significantly greater size of ribbon fields (RF) compared to untreated animals. These data are consistent with hypotheses that pinealocyte function in primates is altered by hormonal imbalances and that the SR participates in this response. RF were positioned in various parts of the cytoplasm and along the plasma membrane. Participation of SR in direct cell-to-cell contacts was suggested by the formation of densities along the plasma membrane. It is postulated that large RF serve as storage organelles and that the formation of RF results from division of pre-existing SR in each field. Reconstructions made from serial thin sections revealed that profiles of RF comprised separate SR that were not folded and sectioned along various planes.

Loss during aging of beta-endorphinergic neurons in the hypothalamus of female C57BL/6J mice

Beta-endorphin (B-EP) content is often reduced in hypothalami of aging rodents. The objective of this study was to determine whether reduced B-EP content is associated with a reduced number of B-EP immunoreactive neurons. Serial coronal sections extending from the caudal hypothalamus through the retrochiasmatic area were examined by quantitative light microscopy in mature (5-6 month) and senescent (24-28 month) mice that had been ovariectomized 1 week earlier and injected with colchicine 24-48 h before sacrifice. Old mice were acyclic. As expected, B-EP immunoreactive cell bodies were restricted to the region of the arcuate nucleus. There was a 35% loss of B-EP immunopositive neurons in old, macroscopically disease-free animals. By contrast, some old animals with pituitary tumors had no loss of B-EP neurons. These results suggest that a subpopulation of B-EP neurons either die or stop synthesizing detectable concentrations of B-EP in aged mice. The basis for the absence of reduced B-EP neurons in some mice with pituitary tumors is unclear, but this observation underscores the importance of distinguishing age-related changes associated with diseases of aging from those that are independent of such diseases.

Ultrastructural distribution of alpha-bungarotoxin binding sites in the suprachiasmatic nucleus of the rat hypothalamus

SummaryThe distribution of (125I) alpha bungarotoxin (α-BTX) binding sites in the suprachiasmatic nucleus (SCN) of the adult female rat was examined by electron-microscopic autoradiography. The ultrastructural distribution of silver grains was analysed by line source, direct point count, and 50% probability circle methods. Real grain distribution was significantly different from that of randomly generated hypothetical grains. Line source analysis demonstrated two populations of sources: one associated with membranes, and one inside neuronal structures. Probability circle analysis of shared grains indicated that membrane-bound-radioactive sources were mainly asssociated with axo-dendritic appositions. Only a small proportion of labeled neuronal interfaces exhibited synaptic differentiations in the plane of section. However, the compartment containing synaptic terminals was the most enriched when comparing real to hypothetical grains. Probability circle analysis of exclusive grains demonstrated that sources that were not associated with neuronal plasma membranes were likely to be within nerve cell bodies and dendrites. It is concluded that the majority of specifically labeled α-BTX binding sites in the SCN is membrane bound, and may be associated with axodendritic synaptic transmission. The presence of a significant proportion of the label in the soma and dendrites of suprachiasmatic neurons 24 h after ventricular infusion suggests that some of the labeled binding sites (junctional or nonjunctional) may be internalized within these two compartments.

Estrogen, the ovary, and neutotransmitters: factors associated with aging

Our studies in the C57BL/6J mouse have been designed to examine the interactions of aging and the ovary, and their mutual effects on neuroendocrine function. In the pituitary, ovarian status and not age determines responsiveness to gonadotropin hormone releasing hormone (GnRH), but estrogen (E2) is an important mediator in CNS changes, and removal of the ovary (OVX) is deleterious to the neuroendocrine hypothalamus. OVX for just six days in young animals results in synaptic loss between noradrenergic terminals and gonadotropin hormone releasing hormone (GnRH) neurons. Long-term OVX, hypothesized to protect against neuroendocrine aging, fails to guard against any studied age-related changes. Some age-related changes occur as early as midlife. Although neuron number remains constant at middle age, opiatergic neurons undergo significant functional changes by producing opiate antagonist peptides. This change appears to be caused by alterations in the prohormone convertases, which cleave propeptide to peptide. Altered peptides may trigger the loss of reproductive capacity. The midlife shift in opiate peptide production is a component of natural developmental processes that begin in the neonate and continue through old age. In the cholinergic system, E2 mediates numbers of cholinergic receptors, cholinergic neurons, and cholinergic-modulated memory systems in both young and old animals. Regardless of age, ovarian steroids, if present at physiologic levels, are beneficial to the neuroendocrine CNS, and long-term deprivation from ovarian-produced factors is deleterious in the systems we have examined. Our studies have shown that deprivation from ovarian steroid hormones in the female appears to be a major factor in the health of the CNS and in events associated with aging.

Aging changes in the β-endorphin neuronal system in the preoptic area of the C57BL/6J mouse: Ultrastructural analysis

In hypothalami of aging rodents, beta-endorphin (beta-EP) neuron number and content are reduced. The objectives of this study were: first, to analyze ultrastructurally the population of neuronal elements in a selected region of the preoptic area (POA) in young and old mice; second, to study the beta-EP neuronal system in the same region to determine whether or not this population remains stable with age. Vibratome sections from the most caudal POA through the diagonal band of Broca were examined by light microscopy and immunocytochemistry in mature, cycling (5-6 months old) and old, acyclic, disease-free (24-26 months old) mice. A subset of beta-EP-like perikarya and associated structures was observed in the periventricular POA. When this subregion was examined at the ultrastructural level, there was a significant decrease in the number of recipient dendrites [3.78 +/- 0.04 SEM/micron 2 young vs. 0.82 +/- 0.03/micron 2 old; p < 0.007, analysis of variance (ANOVA)], but a significant increase in the number of nonmyelinated axons (20.0 +/- 2.6/micron 2 young vs. 26.8 +/- 0.7/micron 2 old; p < 0.05). Immunolabeled terminals that contained a synapse comprised 2.56 +/- 0.08% of all terminals with synapses in young mice but only 0.34 +/- 0.04% in old ones when corrected for surface area examined (p < 0.03). A significant age-related loss was also observed in the nonmyelinated beta-EP-labeled axon population (1.50 +/- 0.10% young vs. 0.40 +/- 0.01% old; p < 0.009, ANOVA). We conclude that there are critical changes in the microenvironment of the POA in old, noncycling female mice that are likely to affect neuron function.