The prodrug DHED selectively delivers 17ß-estradiol to the brain for treating estrogen-responsive disorders.

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17ß-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17ß-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17ß-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

Environmental enrichment protects against the effects of chronic stress on cognitive and morphological measures of hippocampal integrity.

Chronic stress has detrimental effects on hippocampal integrity, while environmental enrichment (EE) has beneficial effects when initiated early in development. In this study, we investigated whether EE initiated in adulthood would mitigate chronic stress effects on cognitive function and hippocampal neuronal architecture, when EE started one week before chronic stress began, or two weeks after chronic stress onset. Adult male Sprague Dawley rats were chronically restrained (6h/d) or assigned as non-stressed controls and subdivided into EE or non-EE housing. After restraint ended, rats were tested on a radial arm water maze (RAWM) for 2-d to assess spatial learning and memory. The first study showed that when EE began prior to 3-weeks of chronic stress, EE attenuated chronic stress-induced impairments in acquisition, which corresponded with the prevention of chronic stress-induced reductions in CA3 apical dendritic length. A second study showed that when EE began 2-weeks after the onset of a 5-week stress regimen, EE blocked chronic stress-induced impairments in acquisition and retention at 1-h and 24-h delays. RAWM performance corresponded with CA3 apical dendritic complexity. Moreover, rats in EE housing (control or stress) exhibited similar corticosterone profiles across weeks, which differed from the muted corticosterone response to restraint by the chronically stressed pair-housed rats. These data support the interpretation that chronic stress and EE may act on similar mechanisms within the hippocampus, and that manipulation of these factors may yield new directions for optimizing brain integrity and resilience under chronic stress or stress related neuropsychological disorders in the adult.

Chronic stress and a cyclic regimen of estradiol administration separately facilitate spatial memory: relationship with hippocampal CA1 spine density and dendritic complexity.

This study investigated the effects of chronic restraint stress and repeated cyclic estradiol pulses on hippocampal CA3 and CA1 dendritic and/or spine morphology and spatial memory in female rats. Sprague-Dawley adult female rats were ovariectomized and then injected over 2 days with 17ß-estradiol (10 µg, s.c.), which was repeated every 4-5 days. While all rats received similar estradiol injection histories, half of the rats were chronically restrained and/or given a final cyclic pulse of estradiol prior to testing on a hippocampal-dependent object placement (OP) task to assess spatial memory. OP testing was performed 2 days after the last restraint session, as well as when the last 2 estradiol pulses best captured the maximal effect on hippocampal CA1 spine density. The data revealed several novel findings: (a) chronic stress or estradiol separately facilitated spatial memory, but did not have the same effects when coadministered, (b) CA1 spine densities negatively correlated with spatial memory, and (c) repeated estradiol pulses failed to prevent stress-induced CA3 dendritic retraction. We also corroborated previous studies showing increased CA1 spine density following estradiol, chronic stress, and behavioral manipulations. The present study uniquely combined chronic stress, repeated estradiol pulses, hippocampal morphology, and behavior within the same animals, allowing for correlational analyses to be performed between CA1 spine morphology and spatial memory. We demonstrate novel findings that chronic stress or estradiol pulses independently facilitate spatial memory, but not when coadministered, and that these effects may involve a balance of CA1 apical spine expression that is independent of CA3 dendritic complexity.

Chronic 17beta-estradiol or cholesterol prevents stress-induced hippocampal CA3 dendritic retraction in ovariectomized female rats: possible correspondence between CA1 spine properties and spatial acquisition.

Chronic stress may have different effects on hippocampal CA3 and CA1 neuronal morphology and function depending upon hormonal status, but rarely are manipulations of stress and gonadal steroids combined. Experiment 1 investigated the effects of chronic restraint and 17beta-estradiol replacement on CA3 and CA1 dendritic morphology and spatial learning in ovariectomized (OVX) female Sprague-Dawley rats. OVX rats were implanted with 25% 17beta-estradiol, 100% cholesterol, or blank silastic capsules and then chronically restrained (6h/d/21d) or kept in home cages. 17beta-Estradiol or cholesterol prevented stress-induced CA3 dendritic retraction, increased CA1 apical spine density, and altered CA1 spine shape. The combination of chronic stress and 17beta-estradiol facilitated water maze acquisition compared to chronic stress + blank implants and nonstressed controls + 17beta-estradiol. To further investigate the interaction between 17beta-estradiol and stress on hippocampal morphology, experiment 2 was conducted on gonadally intact, cycling female rats that were chronically restrained (6h/d/21d), and then euthanized at proestrus (high ovarian hormones) or estrus (low ovarian hormones). Cycling female rats failed to show chronic stress-induced CA3 dendritic retraction at either estrous phase. Chronic stress enhanced the ratio of CA1 basal spine heads to headless spines as found in experiment 1. In addition, proestrous rats displayed increased CA1 spine density regardless of stress history. These results show that 17beta-estradiol or cholesterol protect against chronic stress-induced CA3 dendritic retraction in females. These stress- and 17beta-estradiol-induced morphological changes may provide insight into how dendritic complexity and spine properties contribute to spatial ability.

Chronic stress- and sex-specific neuromorphological and functional changes in limbic structures.

Chronic stress produces sex-specific neuromorphological changes in a variety of brain regions, which likely contribute to the gender differences observed in stress-related illnesses and cognitive ability. Here, we review the literature investigating the relationship between chronic stress and sex differences on brain plasticity and function, with an emphasis on morphological changes in dendritic arborization and spines in the hippocampus, prefrontal cortex, and amygdala. These brain structures are highly interconnected and sensitive to stress and gonadal hormones, and influence a variety of cognitive abilities. Although much less work has been published using female subjects than with male subjects, the findings suggest that the relationship between brain morphology and function is very different between the sexes. After reviewing the literature, we present a model showing how chronic stress influences the morphology of these brain regions and changes the dynamic of how these limbic structures interact with each other to produce altered behavioral outcomes in spatial ability, behavioral flexibility/executive function, and emotional arousal.

Assessment of estradiol influence on spatial tasks and hippocampal CA1 spines: evidence that the duration of hormone deprivation after ovariectomy compromises 17beta-estradiol effectiveness in altering CA1 spines.

Two pulses of 17beta-estradiol (10 microg) are commonly used to increase hippocampal CA1 apical dendritic spine density and alter spatial performance in ovariectomized (OVX) female rats, but rarely are the measures combined. The goal of this study was to use this two-pulse injection protocol repeatedly with intervening wash-out periods in the same rats to: 1) measure spatial ability using different tasks that require hippocampal function and 2) determine whether ovarian hormone depletion for an extended 10-week period reduces 17beta-estradiol's effectiveness in elevating CA1 apical dendritic spine density. Results showed that two injections of 10 microg 17beta-estradiol (72 and 48 h prior to testing and timed to maximize CA1 apical spine density at behavioral assessment) corresponded to improved spatial memory performance on object placement. In contrast, two injections of 5 microg 17beta-estradiol facilitated spatial learning on the water maze compared to rats given two injections of 10 microg 17beta-estradiol or the sesame oil vehicle. Neither 17beta-estradiol dose altered Y-maze performance. As expected, the intermittent two-pulse injection protocol increased CA1 apical spine density, but 10 weeks of OVX without estradiol treatment decreased the effectiveness of 10 microg 17beta-estradiol to increase CA1 apical spine density. Moreover, two pulses of 5 microg 17beta-estradiol injected intermittently failed to alter CA1 apical spine density and decreased basal spine density. These results demonstrate that extended time without ovarian hormones reduces 17beta-estradiol's effectiveness to increase CA1 apical spine density. Collectively, these findings highlight the complex interactions among estradiol, CA1 spine density/morphology, and task requirements, all of which contribute to behavioral outcomes.

Chronic glucocorticoids increase hippocampal vulnerability to neurotoxicity under conditions that produce CA3 dendritic retraction but fail to impair spatial recognition memory.

We previously found that chronic stress conditions producing CA3 dendritic retraction and spatial memory deficits make the hippocampus vulnerable to the neurotoxin ibotenic acid (IBO). The purpose of this study was to determine whether exposure to chronic corticosterone (CORT) under conditions that produce CA3 dendritic retraction would enhance CA3 susceptibility to IBO. Male Sprague Dawley rats were chronically treated for 21 d with CORT in drinking water (400 microg/ml), and half were given daily injections of phenytoin (40 mg/kg), an antiepileptic drug that prevents CA3 dendritic retraction. Three days after treatments stopped, IBO was infused into the CA3 region. Conditions producing CA3 dendritic retraction (CORT and vehicle) exacerbated IBO-induced CA3 damage compared with conditions in which CA3 dendritic retraction was not observed (vehicle and vehicle, vehicle and phenytoin, CORT and phenytoin). Additionally, spatial recognition memory was assessed using the Y-maze, revealing that conditions producing CA3 dendritic retraction failed to impair spatial recognition memory. Furthermore, CORT levels in response to a potentially mild stressor (injection and Y-maze exposure) stayed at basal levels and failed to differ among key groups (vehicle and vehicle, CORT and vehicle, CORT and phenytoin), supporting the interpretations that CORT levels were unlikely to have been elevated during IBO infusion and that the neuroprotective actions of phenytoin were not through CORT alterations. These data are the first to show that conditions with prolonged glucocorticoid elevations leading to structural changes in hippocampal dendritic arbors can make the hippocampus vulnerable to neurotoxic challenges. These findings have significance for many disorders with elevated glucocorticoids that include depression, schizophrenia, Alzheimer's disease, and Cushing's disease.

The effects of chronic stress on hippocampal morphology and function: an evaluation of chronic restraint paradigms.

Chronic restraint stress for 6 h/21 days causes hippocampal CA3 apical dendritic retraction, which parallels spatial memory impairments in male rats. Recent research suggests that chronic immobilization stress for 2 h/10 days induces CA3 dendritic retraction [Vyas, A., Mitra, R., Shankaranarayana Rao, B.S., Chattarji, S., 2002. Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons. J. Neurosci. 22, 6810-6818.] and questions whether CA3 dendritic retraction and spatial memory deficits can be produced sooner than found following 6 h/21 days of restraint stress. Therefore, this study investigated the effects of four different durations of chronic restraint stress (varied by hours/day and total number of days) and the subsequent effects on hippocampal CA3 morphology and spatial memory in the same male Sprague-Dawley rats. The results showed that only rats exposed to the 6 h/21 days restraint paradigm exhibited CA3 apical dendritic retraction, consistent spatial memory deficits, and decreased body weight gain compared to experimental counterparts and controls. While chronically stressing a rat with wire mesh restraint has a physical component, it acts primarily as a psychological stressor, and these findings support the interpretation that chronic psychological stress produces hippocampal-dependent cognitive deficits that are consistent with hippocampal structural changes. Differences in stress effects observed across different studies may be due to rat strain, type of stressor, and housing conditions; however, the current findings support the use of chronic restraint stress, with wire mesh, for 6 h/21 days as a reliable and efficient method to produce psychological stress and to cause CA3 dendritic retraction and spatial memory deficits in male Sprague-Dawley rats.