Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies.

We have shown that 17beta-estradiol exerts profound protective effects against stroke-like ischemic injury in female rats. These effects are evident using physiological levels of estradiol replacement in ovariectomized rats and require hormone treatment prior to the time of injury. The protective actions of estradiol appear to be most prominent in the cerebral cortex, where cell death is not apparent until at least 4 h after the initiation of ischemic injury and where cell death is thought to be apoptotic in nature. Middle-aged rats remain equally responsive to the protective actions of estradiol. The maintenance of responsiveness of the cerebral cortex to the neuroprotective actions of estradiol was unexpected since responsiveness of the hypothalamus to estradiol decreases dramatically by the time animals are middle-aged. We believe that the protective actions of estradiol require the estrogen receptor-alpha, since estradiol does not protect in estrogen receptor-alpha knockout mice. We have also implemented a method of culturing cerebral cortical explants to assess the protective effects of estradiol in vitro. This model exhibits remarkable parallelisms with our in vivo model of brain injury. We have found that 17beta-estradiol decreases the extent of cell death and that this protective effect requires hormone pretreatment. Finally, 17alpha-estradiol, which does not interact effectively with the estrogen receptor, does not protect; and addition of ICI 182,780, an estrogen receptor antagonist, blocks the protective actions of estradiol. We have begun to explore the molecular and cellular mechanisms of estradiol-mediated protection. In summary, our findings demonstrate that estradiol exerts powerful protective effects both in vivo and in vitro and suggest that these actions are mediated by estrogen receptors.

Fos-induction in gonadotropin-releasing hormone neurons receiving vasoactive intestinal polypeptide innervation is reduced in middle-aged female rats.

A hallmark of reproductive aging in rats is a delay in the initiation and peak, and a decrease in the amplitude, of both proestrous and steroid-induced surges of LH and a decrease in the number of GnRH neurons that express Fos during the surge. The altered timing of the LH surge and the decline in Fos expression in GnRH neurons may be due to changes in the rhythmic expression of vasoactive intestinal polypeptide (VIP), a neuropeptide that carries time-of-day information from the circadian pacemaker, located in the suprachiasmatic nuclei (SCN), to GnRH neurons. The goals of our study were to determine if aging alters 1) the innervation of GnRH neurons by VIP and 2) the ability of VIP to activate GnRH neurons by examining the effects of aging on the number of GnRH neurons apposed by VIP fibers and the number of GnRH neurons that receive VIP input that express Fos. Immunocytochemistry for GnRH and VIP; or GnRH, VIP, and Fos was performed on tissue sections collected from young (2-4 mo), regularly cycling females and middle-aged (10-12 mo) females in constant estrus. The number of GnRH neurons, GnRH neurons apposed by VIP fibers, and GnRH neurons that express Fos and apposed by VIP fibers were counted in both age groups. Our results clearly demonstrate that aging does not alter the number of GnRH neurons that receive VIP innervation. However, the number of GnRH neurons that receive VIP innervation and coexpress Fos decreases significantly. We conclude that the age-related delay in the timing of the LH surge is not due to a change in VIP innervation of GnRH neurons, but instead may result from a decreased sensitivity of GnRH neurons to VIP input.

Neuroendocrine influences and repercussions of the menopause.

In summary, the evidence that both the ovary and the brain are key pacemakers in the menopause is compelling. Our appreciation that estrogens are important neurotrophic and neuroprotective factors has grown rapidly. Future studies will allow us to better understand the ensemble of factors that interact to maintain regular reproductive cyclicity and how this precise dynamic balance changes with age. Furthermore, understanding how estrogen exerts trophic and protective actions should lead to its use as an important therapeutic agent in the maintenance of normal neural function during aging and after injury.

Sex differences in the daily rhythm of vasoactive intestinal polypeptide but not arginine vasopressin messenger ribonucleic acid in the suprachiasmatic nuclei.

The timing of the preovulatory surge of LH in female rodents is tightly coupled to the environmental light/dark cycle. This coupling is mediated by the circadian pacemaker located in the suprachiasmatic nuclei (SCN). Studies indicate that vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), which are synthesized in the SCN, transmit circadian information from the SCN to GnRH neurons, thereby regulating the timing of the LH surge. However, to date, the rhythmic expression of these two peptides in the SCN has only been examined in males. The pattern of VIP expression in males is difficult to reconcile with its role in the LH surge. The purpose of the present study was to assess the rhythm of VIP messenger RNA (mRNA) levels in the SCN of female rats under several endocrine conditions. We compared this rhythm to that in males and to AVP mRNA rhythms in all experimental groups. In all groups of females, VIP mRNA levels were rhythmic, with peak expression occurring during the light phase and a nadir occurring during the dark phase. The rhythm was approximately 12 h out of phase compared with that in males. The rhythmic expression of AVP mRNA in the SCN was virtually identical in all groups of animals. Based on these results, we conclude that 1) the rhythm of VIP seen in the SCN of females during the day may serve as a facilitory signal from the SCN to GnRH neurons; 2) the sex-specific pattern of VIP mRNA does not depend on estradiol; and 3) AVP gene expression within the SCN is not sexually differentiated or altered by estradiol.

Aging alters the rhythmic expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin mRNA in the suprachiasmatic nuclei of female rats.

Our laboratory has shown that the ability of the suprachiasmatic nuclei (SCN) to regulate a number of rhythmic processes may be compromised by the time females reach middle age. Therefore, we examined the effects of aging on the rhythmic expression of two neuropeptides synthesized in the SCN, vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), using in situ hybridization. Because both VIP and AVP are outputs of the SCN, we hypothesized that age-related changes in rhythmicity are associated with alterations in the patterns of expression of these peptides. We found that VIP mRNA levels exhibited a 24 hr rhythm in young females, but by the time animals were middle-aged, this rhythm was gone. The attenuation of rhythmicity was associated with a decline in the level of mRNA per cell and in the number of cells in the SCN producing detectable VIP mRNA. AVP mRNA also showed a robust 24 hr rhythm in young females. However, in contrast to VIP, the AVP rhythm was not altered in the aging animals. The amount of mRNA per cell and the number of cells expressing AVP mRNA also was not affected with age. Based on these results we conclude that (1) various components of the SCN are differentially affected by aging; and (2) age-related changes in various rhythms may be attributable to changes in the ability of the SCN to transmit timing information to target sites. This may explain why the deterioration of various rhythmic processes occurs at different rates and at different times during the aging process.

Aging of the female reproductive system: a window into brain aging.

The menopause marks the permanent end of fertility in women. It was once thought that the exhaustion of ovarian follicles was the single, most important explanation for the transition to the menopause. Over the past decade, this perception has gradually changed with the realization that there are multiple pacemakers of reproductive senescence. We will present evidence that lends credence to the hypothesis that the central nervous system is a critical pacemaker of reproductive aging and that changes at this level contribute to the timing of the menopause. Studies demonstrate that an increasing de-synchronization of the temporal order of neuroendocrine signals may contribute to the accelerated rate of follicular loss that occurs during middle age. We suggest that the dampening and destabilization of the precisely orchestrated ultradian, circadian, and infradian neural signals lead to miscommunication between the brain and the pituitary-ovarian axis. This constellation of hypothalamic-pituitary-ovarian events leads to the inexorable decline of regular cyclicity and heralds menopausal transition.

In vivo antisense antagonism of vasoactive intestinal peptide in the suprachiasmatic nuclei causes aging-like changes in the estradiol-induced luteinizing hormone and prolactin surges.

In mammals, the suprachiasmatic nuclei (SCN) regulate the timing of LH surges. Recent evidence suggests that vasoactive intestinal peptide (VIP), an abundantly expressed neuropeptide of the SCN, communicates time of day information from the SCN to GnRH neurons. VIP levels in the SCN decrease with age and may be responsible for alterations in LH surges that become apparent in middle-aged rats. We wished to determine whether suppression of VIP synthesis, through antisense oligonucleotides (oligos) directed at the SCN, results in 1) selective suppression of VIP levels in the SCN and 2) aging-like changes in the secretion of LH and PRL. To test the specificity of antisense oligo treatment, rats were ovariectomized and treated with estradiol. Antisense or control random oligos were infused into the peri-SCN region through stereotaxically placed bilateral cannulas. Beginning at lights off, rats were maintained in constant dim red illumination throughout the remainder of the experiment. They were killed at specific times, brains were microdissected, and VIP concentrations in the SCN, paraventricular nuclei, and cortex were assayed. As a control for the specificity of antisense VIP treatment, we monitored the levels of arginine vasopressin in the SCN. To test the effects of antisense treatment on the pattern of plasma LH and PRL secretion, blood samples were collected from atrial catheters from 1200-2000 h, and plasma samples were assayed for LH and PRL. The results indicate that the effects of antisense treatment were discrete, as they suppressed VIP concentrations in the SCN, but had no effect on VIP concentrations in the paraventricular nuclei or cortex or on arginine vasopressin concentrations in the SCN. Peak LH levels during the surge were delayed and attenuated in antisense-treated animals compared to random oligo-treated control rats in a manner strikingly similar to that observed previously in middle-aged rats. Likewise, PRL, which was unaffected in middle-aged rats, was also unaffected by targeted suppression of VIP. In summary, our findings clearly demonstrate that antisense VIP oligos suppress VIP levels in the SCN and do not affect peptide concentrations in other regions of the brain or other neuropeptides in the SCN. Further, we show that suppression of a single neuropeptide in the SCN can mimic the effects of age on the estradiol-induced surges of LH and PRL. These data support a central role for suprachiasmatic VIP in the regulation of the LH surge and suggest that age-related perturbations in the integrity of this axis may account for alterations in the pattern of LH secretion observed during middle age.

Acute effects of antisense antagonism of a single peptide neurotransmitter in the circadian clock.

The circadian clock that resides in the suprachiasmatic nucleus (SCN) of the hypothalamus is the major neural pacemaker driving most 24-h rhythms in mammals. Several neurotransmitter peptides are synthesized within this nucleus and communicate rhythmically with other cells in the SCN as well as with cells in other regions of the brain. At the present time, little is known about their role in regulating outputs of the clock. We demonstrate that antisense oligodeoxynucleotides corresponding to the NH2-terminus and the translation start site of vasoactive intestinal peptide (VIP) mRNA infused into the suprachiasmatic region of rats temporarily abolishes the circadian rhythm of corticosterone secretion without influencing stress-related corticosterone secretion in the same animals. Levels of VIP peptide are suppressed 30% on the second day after antisense treatment. These results indicate that a single neuropeptide transmitter in the circadian clock may serve a distinct role in the control of specific circadian rhythms.

Effect of lactation on hypothalamic preproenkephalin gene expression.

Enkephalin appears to modulate several aspects of reproductive function in female rats. The purpose of this study was to determine if lactation influences preproenkephalin gene expression in one or more hypothalamic nuclei known to be involved in maternal or reproductive behavior and prolactin secretion. Lactating rats were killed on day 3 (LAC 3) or day 10 (LAC 10) of lactation. Controls consisted of regular 4-day cycling rats that were killed on diestrous day 1, with 9 to 12 females per group. We used in situ hybridization histochemistry to assess preproenkephalin gene expression in individual cells in the medial preoptic nucleus, anterior, medial and posterior arcuate nucleus, magnocellular and parvocellular aspects of paraventricular nucleus, and ventromedial nucleus. Preproenkephalin mRNA in the anterior arcuate nucleus increased to reach significance (P < 0.05) at day 10 of lactation. Levels in the medial arcuate nucleus increased significantly (P < 0.001) by day 3 of lactation (LAC 3) and remained elevated on day 10 (LAC 10). No significant differences between lactating and control rats were detected in preproenkephalin mRNA levels in the posterior arcuate nucleus, medial preoptic nucleus or in the ventromedial nucleus. Substantial levels of preproenkephalin mRNA were found in the paraventricular nucleus, particularly in a limited region of the magnocellular portion. However, these levels did not change with lactation. These data provide evidence for differential regulation of the preproenkephalin gene during lactation. This change may contribute to lactational hyperprolactinemia and suppressed GnRH secretion, leading to reproductive acyclicity.

A period (per)-like protein exhibits daily rhythmicity in the suprachiasmatic nuclei of the rat.

The period (per) gene of Drosophila melanogaster is considered an important biological clock gene, since it regulates multiple behavioral rhythms. Per mRNA and protein exhibit circadian rhythms in the fruitfly brain and these rhythms appear to influence each other through a feedback loop. More recently, using the same antibody as was used in the Drosophila studies, PER-like proteins were detected in the suprachiasmatic nuclei (SCN) of male rats. This region of the brain is considered to be a major neural circadian pacemaker in mammals. The purpose of this study was to confirm that PER-like proteins are detectable in the SCN of female rats and to determine whether PER-like proteins exhibit a circadian rhythm. Female rats were killed at several times of day under both light/dark and constant conditions. Using the same anti-PER antibody in Western blots with Enhanced Chemiluminescence (Western-ECL) detection, the levels of the PER-like proteins were quantified in the SCN and cerebral cortex. The antibody identified a doublet band of approximately 170-160 kDa and a single band at 115 kDa. Of the three PER-like proteins only the largest exhibited a daily rhythm in the SCN, which peaked in the middle of the dark and attained its nadir around lights off; levels during the light were intermediate with a tendency towards a second drop around lights on. This rhythm did not persist under constant dim red light.(ABSTRACT TRUNCATED AT 250 WORDS)