Stroke injury in rats causes an increase in activin A gene expression which is unaffected by oestradiol treatment.

Activins are members of the transforming growth factor-beta superfamily that exert neurotrophic and neuroprotective effects on various neuronal populations. To determine the possible function of activin in stroke injury, we assessed which components of the activin signalling pathway were modulated in response to middle cerebral artery occlusion (MCAO). Furthermore, because oestradiol replacement protects against MCAO-induced cell death, we explored whether oestradiol replacement influences activin gene expression. Female Sprague-Dawley rats underwent permanent MCAO and the expression of activins and their corresponding receptors was determined by semiquantitative reverse transcriptase-polymerase chain reaction at 24 h after onset of ischaemia. We observed up-regulation of activin betaA and activin type I receptor A mRNA in response to injury. Dual-label immunocytochemistry followed by confocal z-stack analysis showed that the activin A expressing cells comprised neurones. Next, we monitored the time course of activin betaA mRNA expression in oestradiol- or vehicle-treated rats at 4, 8, 16 and 24 h after MCAO via in situ hybridisation. Starting at 4 h after injury, activin betaA mRNA was up-regulated in cortical and striatal areas in the ipsilateral hemisphere. Activin betaA mRNA levels in the cortex increased dramatically with time and were highest at 24 h after the insult, and oestradiol replacement did not influence this increase.

Estrogen therapy: does it help or hurt the adult and aging brain? Insights derived from animal models.

Hormone therapy and estrogen therapy in postmenopausal women have been thought to ameliorate cognitive dysfunction and decrease the risk and/or progress of neurodegenerative conditions such as Alzheimer's disease and stroke. Furthermore, estrogens have been shown to exert neuroprotective actions in a variety of in vitro and in vivo models of brain injury. However, the findings of the Women's Health Initiative have made us re-evaluate these assumptions. Our laboratory has shown that physiological levels of estradiol attenuate ischemic brain injury in young and middle-aged female rats. We have begun to probe the cellular and molecular mechanisms that underlie these novel non-reproductive actions of this steroid. Our findings demonstrate that in both young and aging rats, treatment with physiological concentrations of estradiol decreases ischemic injury by almost 50%, compared with oil-treated controls. Additionally, our data suggest that estradiol acts by altering the expression of genes that suppress apoptosis and enhance survival in the penumbral region of the infarct. These observations demonstrate that estrogen therapy protects against stroke-related injury in young and aging female rats and strongly suggest that middle-aged animals remain responsive to the protective actions of estradiol. Furthermore, they suggest that estrogen therapy protects against cell death by influencing the expression of genes that suppress apoptotic cell death pathways.

Glutamic acid decarboxylase 67 (GAD67) gene expression in discrete regions of the rostral preoptic area change during the oestrous cycle and with age.

Gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, is important to the timing and amplitude of the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) surge on pro-oestrus. Data suggest that GABA input in the preoptic area must decrease for a normal LH surge to occur in young rats. We have previously found that ageing alters the timing and amplitude of the LH surge. Therefore, this study focused on changes in GAD(67) gene expression, a reflection of GABA synthesis, in two regions of the rostral preoptic area, the organum vasculosum of the lamina terminalis (OVLT) and the anteroventral periventricular nucleus (AVPV) during the oestrous cycle and with age. We assessed the possibility that the expression of GAD(67) mRNA in these regions displays time-related and age-dependent changes on pro-oestrus. Our results demonstrate that, with age, overall expression of GAD(67) mRNA decreases in the area surrounding the OVLT and in the AVPV. Young rats display a diurnal rhythm in GAD(67) mRNA in both regions. GAD(67) mRNA expression is high during the early morning hours of pro-oestrus and then declines around the time of the GnRH-induced LH surge. In addition, the diurnal rhythm disappears in the AVPV and is attenuated in the area surrounding the OVLT of middle-aged proestrous rats. These findings suggest that a loss of rhythmicity in GAD(67) gene expression and maintenance of inhibitory tone on proestrous afternoon may alter the timing and amplitude of the LH surge, as previously observed in middle-aged rats.

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.

The 'menopause' and the aging brain: causes and repercussions of hypoestrogenicity.

We are beginning to recognize that the regulation of reproductive senescence in females and its consequences are more complex than originally thought. We now realize that the brain plays an important role in the transition to infertility in rodents and perhaps contributions to the perimenopausal transition in women. Furthermore, the absence of estrogens leads to changes in many physiological systems. Thus, it is becoming clear that it is important to understand the broad impact of the prolonged hypoestrogenic state that characterizes the menopause.

Chronic smoking enhances tachykinin synthesis and airway responsiveness in guinea pigs.

This study tests the hypothesis that the bronchial hyperreactivity induced by chronic cigarette smoke (CS) exposure involves the increased expression and release of tachykinins and calcitonin gene-related peptide (CGRP) from afferent nerve fibers innervating the airways. In guinea pigs chronically exposed to CS (20 min twice daily for 14-17 d), peak response in total lung resistance to capsaicin (1.68 microg/kg, intravenously) was significantly greater than that evoked by the same dose of capsaicin in control (air-exposed) animals. This augmented response in CS-exposed animals was abolished after treatment with CP-99994 and SR-48968, the neurokinin (NK)-1 and NK-2 receptor antagonists, suggesting the involvement of tachykinins in chronic CS-induced airway hyperresponsiveness (AHR). Further, substance P (SP)-like immunoreactivity (LI) and CGRP-LI in the airway tissue were significantly greater in the CS animals than in the control animals. Finally, beta-preprotachykinin (PPT, a splice variant from the PPT A gene encoding tachykinins including SP and NKA) messenger RNA levels as measured by in situ hybridization histochemistry displayed a significant increase in jugular ganglion neurons but not in dorsal root or nodose ganglion neurons. These data suggest that chronic CS-induced AHR is related to an increase in SP synthesis and release in jugular ganglion neurons innervating the lungs and airways.

Orbital response indicates nasal pungency: analysis of biomechanical strain on the skin.

Stimulation of the human nasal passage with pungent vapor elicits motor responses in a zone around the eye. This investigation addressed whether quantification of such responses, particularly activity of the orbicularis oculi muscle, could yield a sensitive index of nasal pungency. We placed an array of small, high-contrast targets just beneath the lower eyelid and videotaped their movement to capture deformation of the skin atop the orbicularis oculi during 3 s stimulation with pungent concentrations of ethyl acetate. Eleven subjects participated. Analysis of the movements served to determine mechanical strain, which yielded a single index that we termed 'maximum strain'. This increased with concentration of the vapor and with time during and just after stimulation. Comparison with psychophysical data showed that the strain became evident at concentrations just detectable as pungent. Maximum strain measured on the skin shows promise as an objective index of pungency.

Parallel declines in Fos activation of the medial anteroventral periventricular nucleus and LHRH neurons in middle-aged rats.

The middle-age decline in reproductive function is manifested by reduced LHRH release, resulting in a decreased magnitude and delay of onset of the LH surge. Earlier studies suggested that the reductions in LHRH neural activation in middle-aged rats resulted from deficits in the afferent drive to the LHRH neurons. One critical afferent to the LHRH neurons lies in the anteroventral periventricular preoptic area (AVPv) nucleus. The neurons of the medial AVPv are synchronously activated to express Fos with LHRH neurons at the time of an LH surge in young adult animals. The present study examined whether, in middle age, reductions in the activation of AVPv neurons accompany the reduction in Fos activation in LHRH neurons. Young (3- to 4-month-old) and middle-aged (10- to 12-month-old) spontaneously cycling and ovariectomized steroid-replaced rats were killed during peak and early descending phase of the LH surge, and their brains were examined for Fos in LHRH and AVPv neurons. Young animals had a characteristic increase in Fos expression in both LHRH and AVPv neurons. In middle-aged rats, the proportion of LHRH neurons expressing Fos at the time of an LH surge was reduced by approximately 50%, irrespective of whether surges were spontaneous or induced by exogenous steroids. A similar reduction in the number of Fos+ cells (by approximately 50%) was noted in the medial AVPv. Linear regression analysis of the relationship between the extent of Fos activation in LHRH and AVPv neurons revealed a strong positive correlation (r(2) = 0.66; P < 0.01), suggesting that changes in the AVPv's drive to LHRH neurons underlie the decrease in LHRH activity in middle age. A second series of experiments examined whether decreased input from the AVPv could account for reduced Fos activation in LHRH neurons seen in middle-aged animals. When the medial AVPv was lesioned, LHRH neurons failed to express Fos on the side ipsilateral to the lesion. Animals with lesioned medial AVPv also had significantly lower LH values than animals with an intact medial AVPv. Taken together, these data suggest that a principal deficit in middle-aged rats is the ability of the medial AVPv to stimulate LHRH neurons.

Neurotensin gene expression increases during proestrus in the rostral medial preoptic nucleus: potential for direct communication with gonadotropin-releasing hormone neurons.

Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.

Localization of kappa opioid receptors in oxytocin magnocellular neurons in the paraventricular and supraoptic nuclei.

It has been demonstrated previously that kappa opioid receptor agonists, such as dynorphin, inhibit oxytocin secretion in the rat. To determine whether kappa agonists act directly on oxytocin-containing magnocellular neurons to inhibit hormone secretion, we utilized immunofluorescence to examine the cellular localization of kappa opioid receptors in the rat paraventricular and supraoptic nuclei. kappa Opioid receptor immunoreactivity co-localized with oxytocin-containing cell bodies, their axons and axon terminals. Thus, our results suggest that kappa opioid receptor agonists can exert direct inhibitory actions on oxytocin magnocellular neurons.

Effects of estrogen deficiency on brain function. Implications for the treatment of postmenopausal women.

A growing body of evidence suggests that postmenopausal estrogen deficiency accelerates brain aging and increases the risk of various neurodegenerative processes, including Alzheimer's disease. Recent preclinical and clinical studies have indicated that estrogen has positive effects on brain homeostasis by preserving neural plasticity and the neurotransmitter pathways involved in learning, memory, and balance. In this article, Dr Birge and his coauthors address the effects of estrogen on brain function and discuss their implications for the use of selective estrogen receptor modulators, particularly tamoxifen and raloxifene, in postmenopausal women.

Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine.

BACKGROUND: Human immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness. To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat. RESULTS: Acute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors. CONCLUSION: Our observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

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.

Minireview: neuroprotective effects of estrogen-new insights into mechanisms of action.

An accumulating body of evidence clearly establishes that estradiol is a potent neuroprotective and neurotrophic factor in the adult: it influences memory and cognition, decreases the risk and delays the onset of neurological diseases such as Alzheimer's disease, and attenuates the extent of cell death that results from brain injuries such as cerebrovascular stroke and neurotrauma. Thus, estradiol appears to act at two levels: 1) it decreases the risk of disease or injury; and/or 2) it decreases the extent of injury incurred by suppressing the neurotoxic stimulus itself or increasing the resilience of the brain to a given injury. During the past century, the average life span of women has increased dramatically, whereas the time of the menopause has remained essentially constant. Thus, more women will live a larger fraction of their lives in a postmenopausal, hypoestrogenic state than ever before. Clearly, it is critical for us understand the circumstances under which estradiol exerts protective actions and the cellular and molecular mechanisms that underlie these novel, nonreproductive actions.

Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury.

Estradiol protects against brain injury, neurodegeneration, and cognitive decline. Our previous work demonstrates that physiological levels of estradiol protect against stroke injury and that this protection may be mediated through receptor-dependent alterations of gene expression. In this report, we tested the hypothesis that estrogen receptors play a pivotal role in mediating neuroprotective actions of estradiol and dissected the potential biological roles of each estrogen receptor (ER) subtype, ER alpha and ER beta, in the injured brain. To investigate and delineate these mechanisms, we used ER alpha-knockout (ER alpha KO) and ER beta-knockout (ER beta KO) mice in an animal model of stroke. We performed our studies by using a controlled endocrine paradigm, because endogenous levels of estradiol differ dramatically among ER alpha KO, ER beta KO, and wild-type mice. We ovariectomized ER alpha KO, ER beta KO, and the respective wild-type mice and implanted them with capsules filled with oil (vehicle) or a dose of 17 beta-estradiol that produces physiological hormone levels in serum. One week later, mice underwent ischemia. Our results demonstrate that deletion of ER alpha completely abolishes the protective actions of estradiol in all regions of the brain; whereas the ability of estradiol to protect against brain injury is totally preserved in the absence of ER beta. Thus, our results clearly establish that the ER alpha subtype is a critical mechanistic link in mediating the protective effects of physiological levels of estradiol in brain injury. Our discovery that ER alpha mediates protection of the brain carries far-reaching implications for the selective targeting of ERs in the treatment and prevention of neural dysfunction associated with normal aging or brain injury.

Estrogens: trophic and protective factors in the adult brain.

Our appreciation that estrogens are important neurotrophic and neuroprotective factors has grown rapidly. Although a thorough understanding of the molecular and cellular mechanisms that underlie this effect requires further investigation, significant progress has been made due to the availability of animal models in which we can test potential candidates. It appears that estradiol can act via mechanisms that require classical intracellular receptors (estrogen receptor alpha or beta) that affect transcription, via mechanisms that include cross-talk between estrogen receptors and second messenger pathways, and/or via mechanisms that may involve membrane receptors or channels. This area of research demands attention since estradiol may be an important therapeutic agent in the maintenance of normal neural function during aging and after injury.

Neuroprotective effects of estradiol in middle-aged female rats.

Estrogen replacement therapy in postmenopausal women ameliorates cognitive dysfunction and decreases the risk and/or severity of neurodegenerative conditions such as Alzheimer's disease and stroke. Furthermore, estradiol exerts neuroprotective effects in a variety of in vitro and in vivo models of brain injury. We have previously shown that physiological levels of estradiol attenuate ischemic brain injury in young female rats. However, neurodegenerative events occur more frequently in elderly women who are chronically hypoestrogenic. Therefore, we investigated whether aging rats remain responsive to the neuroprotective actions of estradiol. Young (3-4 months) and middle-aged (9-12 months) rats were ovariectomized and treated for 1 week with estradiol before middle cerebral artery occlusion (MCAO). Regional cerebral blood flow was monitored in some animals at the time of injury. Brains were collected 24 h after MCAO and infarct volume was analyzed. Our data demonstrate that in both young and aging rats, low and high physiological doses of estradiol decrease ischemic injury by almost 50%, compared with oil-treated controls. Additionally, our data suggest that estradiol acts in both age groups via blood flow-independent mechanisms, as basal and postinjury blood flow was equivalent between estradiol- and oil-treated young and aging rats. These data demonstrate that replacement with physiological levels of estradiol protects against stroke-related injury in young and aging female rats and strongly suggest that older animals remain responsive to the protective actions of estradiol.

Localization of the VIP2 receptor protein on GnRH neurons in the female rat.

In mammals, the timing and occurrence of the preovulatory LH surge critically depends on the proper functioning of the suprachiasmatic nucleus (SCN). Recent evidence suggests that vasoactive intestinal polypeptide (VIP) conveys time of day information from the SCN to GnRH neurons. However, it is not completely clear whether this action is exerted directly at the level of the GnRH neuron. To determine if GnRH neurons are direct targets for VIP, triple-label immunofluorescence was utilized to simultaneously localize GnRH, VIP and VIP2 receptor protein. The present results demonstrate that about 40% of all GnRH neurons analyzed contain VIP2 receptor immunoreactivity and that VIP-containing processes were seen in close apposition to a significant number of VIP2 receptor-positive GnRH neurons. GnRH neurons that exhibit immunoreactivity for the VIP2 receptor are located predominantly in the OVLT region and the rostral preoptic area. In the median eminence, where the majority of GnRH neurons terminate, VIP2 receptor immunoreactivity was absent. In summary, these findings indicate that VIP can communicate directly with GnRH neurons.

Estradiol protects against ischemic brain injury in middle-aged rats.

Several clinical studies suggest that estradiol acts as a potent growth and protective factor in the adult brain. Postmenopausal women experience permanent hypoestrogenicity and suffer from increased risk of brain injury associated with neurodegenerative diseases such as stroke and Alzheimer's disease. Estrogen replacement therapy appears to decrease the risk and severity of these neurodegenerative conditions. Studies using animal models have shown that estradiol exerts similar effects in rodents and can enhance cell survival and induce synaptic plasticity. Therefore, we undertook studies to assess whether estradiol treatment can decrease brain injury and cell death induced by an experimental model of ischemia and whether aging animals remain responsive to the protective effects of estradiol. We will review results from recent studies that demonstrate that 1) in young animals, estrogens exert profound protective effects against ischemic brain injury induced by cerebral artery occlusion and 2) the response of aging animals has been tested with varying results. We will discuss and compare our experimental findings that utilize a permanent cerebral artery occlusion model and physiological levels of estradiol replacement therapy in young and middle-aged rats with those of previous studies. These observations provide important insights into the potential protective actions of estrogen replacement therapy on age- and disease-related processes in the brain.