Neuroestrogen-Dependent Transcriptional Activity in the Brains of ERE-Luciferase Reporter Mice following Short- and Long-Term Ovariectomy.

Previous work has demonstrated that estrogen receptors are transcriptionally active in the absence of ovarian estrogens. The current work aims to determine whether brain-derived estrogens influence estrogen receptor-dependent transcription after short- or long-term loss of ovarian function. Experiments were conducted using estrogen response element (ERE)-Luciferase reporter mice, which express the gene for luciferase driven by consensus ERE, allowing for the quantification of ERE-dependent transcription. Brain regions examined were hippocampus, cortex, and hypothalamus. In Experiment 1, short-term (10 d) ovariectomy had no impact on ERE-dependent transcription across brain regions compared with sham surgery. In Experiment 2, chronic intracerebroventricular administration of the aromatase inhibitor letrozole significantly decreased transcriptional activity in 10-d-old ovariectomized mice across brain regions, indicating that the sustained transcription in short-term ovariectomized mice is mediated at least in part via actions of neuroestrogens. Additionally, intracerebroventricular administration of estrogen receptor antagonist ICI-182,780 blocked transcription in 10-d-old ovariectomized mice across brain regions, providing evidence that sustained transcription in ovariectomized mice is estrogen receptor dependent. In Experiment 3, long-term (70 d) ovariectomy significantly decreased ERE-dependent transcription across brain regions, though some residual activity remained. In Experiment 4, chronic intracerebroventricular letrozole administration had no impact on transcription in 70 d ovariectomized mice across brain regions, indicating that the residual ERE-dependent transcription in long-term ovariectomized mice is not mediated by neuroestrogens. Overall, the results indicate that ERE-dependent transcription in the brain continues after ovariectomy and that the actions of neuroestrogens contribute to the maintenance of ERE-dependent transcription in the brain following short-term, but not long-term, loss of ovarian function.

Organizational effects of testosterone on learning strategy preference and muscarinic receptor binding in prepubertal rats.

Prior to puberty, male rats, but not female rats, prefer a striatum-based stimulus-response learning strategy rather than a hippocampus-based place strategy on a water maze task that can be solved using either strategy. Neurochemically, learning strategy preference has been linked to the ratio of cholinergic muscarinic receptor binding in the hippocampus relative to the striatum, with lower ratios displayed by males compared to females and by stimulus-response learners compared to place learners. Sex differences in a variety of different behaviors are established by the organizational influence of testosterone on brain development. Therefore, the current study investigated the potential organizational effects of neonatal testosterone on learning strategy preference and the hippocampus:striatum ratio of muscarinic receptor binding in prepubertal male and female rats. Similar to vehicle-treated control males, prepubertal females treated with testosterone propionate on the first two days of life preferred a stimulus-response strategy on a dual-solution water maze task. Conversely, vehicle-treated prepubertal females were more likely to use a place strategy. Consistent with previous findings, the hippocampus:striatum ratio of muscarinic receptor binding was lower in rats preferring a stimulus-response strategy compared to those using a place strategy and lower in control males compared to control females. However, the hippocampus:striatum ratio was not reversed by neonatal testosterone treatment of females as predicted. The current study is the first to show that sex differences in how a navigational task is learned prior to puberty is impacted by the presence of testosterone during vulnerable periods in brain development.

Long- but not short-term estradiol treatment induces renal damage in midlife ovariectomized Long-Evans rats.

Clinical recommendations limit menopausal hormone therapy to a few years, yet the impact of a shorter treatment duration on cardiovascular health is unknown. We hypothesized that both short- and long-term estradiol (E2) treatment exerts positive and lasting effects on blood pressure, vascular reactivity, and renal health. This study was designed to mimic midlife menopause, followed by E2 treatment, that either followed or exceeded the current clinical recommendations. Female Long-Evans retired breeders were ovariectomized (OVX) at 11 mo of age and randomized into three groups: 80-day (80d) vehicle (Veh>Veh), 40-day (40d) E2 + 40d vehicle (E2>Veh), and 80d E2 (E2>E2). In comparison to Veh>Veh, both the E2>Veh and E2>E2 groups had lower systolic blood pressure and enhanced mesenteric relaxation in response to estrogen receptor-α stimulation. Despite the reduced blood pressure, E2>E2 induced renal and cardiac hypertrophy, reduced glomerular filtration, and increased proteinuria. Interestingly, kidneys from E2>Veh rats had significantly fewer tubular casts than both of the other groups. In conclusion, long-term E2 lowered blood pressure but exerted detrimental effects on kidney health in midlife OVX Long-Evans rats, whereas short-term E2 lowered blood pressure and reduced renal damage. These findings highlight that the duration of hormone therapy may be an important factor for renal health in aging postmenopausal women.

Evidence for Ligand-Independent Activation of Hippocampal Estrogen Receptor-α by IGF-1 in Hippocampus of Ovariectomized Rats.

In the absence of ovarian estrogens, increased levels of estrogen receptor (ER)α in the hippocampus are associated with improvements in cognition. In vitro evidence indicates that under conditions of low estrogen, growth factors, including Insulin-Like Growth Factor 1 (IGF-1), can activate ERα and regulate ERα-mediated transcription through mechanisms that likely involve modification of phosphorylation sites on the receptor. The goal of the current work was to investigate a role for IGF-1 in ligand-independent activation of ERα in the hippocampus of female rats. Ovariectomized rats received a single intracerebroventricular infusion of IGF-1 and hippocampi were collected 1 or 24 hours later. After 1 h, IGF-1 increased hippocampal levels of phosphorylated ERα at serine 118 (S118) as revealed by Western blotting. Coimmunoprecipitation revealed that at 1 hour after infusion, IGF-1 increased association between ERα and steroid receptor coactivator 1, a histone acetyltransferase that increases transcriptional activity of phosphorylated ERα. IGF-1 infusion increased levels of the ERα-regulated proteins ERα, choline acetyltransferase, and brain-derived neurotrophic factor in the hippocampus 24 hours after infusion. Results indicate that IGF-1 activates ERα in ligand-independent manner in the hippocampus via phosphorylation at S118 resulting in increased association of ERα with steroid receptor coactivator 1 and elevation of ER-regulated proteins. To our knowledge, these data are the first in vivo evidence of ligand-independent actions of ERα and provide a mechanism by which ERα can impact memory in the absence of ovarian estrogens.

Effect of botulinum-A toxin injection into bulbospongiosus muscle on ejaculation latency in male rats.

INTRODUCTION: Premature ejaculation (PE) is the most common male sexual dysfunction. A variety of pharmacotherapeutic strategies have been employed to treat men suffering with lifelong PE. However, there are currently no pharmaceuticals approved by the U.S. Food and Drug Administration specifically designed for PE treatment. AIM: Given that the bulbospongiosus muscle is involved in the ejaculatory reflex in both humans and rodents and that local administration of botulinum-A can abolish muscle contractions, the current study examined the effect of injection of botulinum-A toxin into the bulbospongiosus muscle on the ejaculatory latency of male rats. METHODS: After screening for normal sexual activity with sexually receptive female rats, 33 sexually experienced male Long-Evans rats (Harlan Laboratories, Indianapolis, IN, USA) underwent an additional four pretreatment sexual exposures over the course of the following week, during which all components of sexual behavior were video recorded by trained observers. On the day after their fourth experience, rats were anesthetized and received a single injection of either 0.5 unit (n = 11) or 1 unit (n = 11) of botulinum-A toxin or saline vehicle (n = 11). Botulinum-A toxin was dissolved in 0.1 mL of saline vehicle and injected bilaterally into the bulbospongiosus muscle by the percutaneous route. Beginning 2 days after treatment, sexual behaviors were reexamined over the course of the following week on four separate occasions. MAIN OUTCOME MEASURES: The latency to achieve ejaculation, and the frequencies and latencies of mounts and intromissions were video recorded by trained observers in a blinded fashion. RESULTS: Relative to pretreatment measurements, bilateral injection of saline vehicle into the bulbospongiosus muscle did not affect ejaculation latencies. However, rats treated with either 0.5 or 1 unit of botulinum-A toxin exhibited significantly longer latencies to achieve ejaculation relative to pretreatment performance. Of note, botulinum-A toxin did not affect the ability to achieve mounts, intromissions, or ejaculation. CONCLUSIONS: These results demonstrate that botulinum-A toxin injection into the bulbospongiosus muscle is a safe and effective treatment that extends ejaculatory latency in rats without affecting the ability to engage in sexual activity or achieve ejaculation. Further studies are required to evaluate this therapeutic concept in PE patients.

Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats.

In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the α or ß isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1 µg) of estradiol 48 h and 24 h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48 h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25 µg) 48 h and 24 h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol.

Testosterone modulates spatial recognition memory in male rats.

A growing body of research indicates that testosterone influences spatial cognition in male rats; however, the overwhelming majority of studies have been conducted on tasks motivated by either food deprivation or water escape. The hippocampus-dependent version of the Y-maze task, which characterizes spatial recognition memory, capitalizes on the propensity of rats to gravitate toward novel spatial environments and is not contingent upon either appetite or the stress associated with water escape, two factors also affected by testosterone. Accordingly, the aim of the current study was to examine the effects of orchidectomy and subsequent testosterone treatment on spatial recognition memory. Orchidectomy did not impact spatial recognition memory when the delay between the information and retention trials of the Y-maze task was 24h. Alternatively, on the second Y-maze task, which featured a 48-h delay between trials, orchidectomy reduced, and treatments that produced higher levels of testosterone restored, preference for the arm associated with the novel spatial environment. Importantly, there were no differences in activity levels as a function of orchidectomy or testosterone treatment on either of the two tasks. Consistent with previous findings, orchidectomy attenuated, and testosterone treatment restored, both body weight gain and the relative weight of the androgen-sensitive ischiocavernosus muscle, which confirmed the efficacy of orchidectomy and testosterone treatments on physiological outcomes. Therefore, testosterone influenced spatial cognition on a task that minimized the influence of non-mnemonic factors and took advantage of the innate preference of rodents to seek out novel spatial environments.

Biological sex influences learning strategy preference and muscarinic receptor binding in specific brain regions of prepubertal rats.

According to the theory of multiple memory systems, specific brain regions interact to determine how the locations of goals are learned when rodents navigate a spatial environment. A number of factors influence the type of strategy used by rodents to remember the location of a given goal in space, including the biological sex of the learner. We recently found that prior to puberty male rats preferred a striatum-dependent stimulus-response strategy over a hippocampus-dependent place strategy when solving a dual-solution task, while age-matched females showed no strategy preference. Because the cholinergic system has been implicated in learning strategy and is known to be sexually dimorphic prior to puberty, we explored the relationship between learning strategy and muscarinic receptor binding in specific brain regions of prepubertal males and female rats. We confirmed our previous finding that at 28 days of age a significantly higher proportion of prepubertal males preferred a stimulus-response learning strategy than a place strategy to solve a dual-solution visible platform water maze task. Equal proportions of prepubertal females preferred stimulus-response or place strategies. Profiles of muscarinic receptor binding as assessed by autoradiography varied according to strategy preference. Regardless of biological sex, prepubertal rats that preferred stimulus-response strategy exhibited lower ratios of muscarinic receptor binding in the hippocampus relative to the dorsolateral striatum compared to rats that preferred place strategy. Importantly, much of the variance in this ratio was related to differences in the ventral hippocampus to a greater extent than the dorsal hippocampus. The ratios of muscarinic receptors in the hippocampus relative to the basolateral amygdala also were lower in rats that preferred stimulus-response strategy over place strategy. Results confirm that learning strategy preference varies with biological sex in prepubertal rats with males biased toward a stimulus-response strategy, and that stimulus-response strategy is associated with lower ratios of muscarinic binding in the hippocampus relative to either the striatum or amygdala.

Reactivation of an aversive memory modulates learning strategy preference in male rats.

Reminders of an aversive event adversely impact retrieval of hippocampus-dependent memories and exacerbate stress-induced levels of anxiety. Interestingly, stress and anxiety shift control over learning away from the hippocampus and toward the striatum. The aims of the current study were to determine whether spatial memory and learning strategy are impacted by reminders of a stressor. Adult male Long-Evans rats (N = 47) were subjected to an inhibitory avoidance (IA) training trial in which 32 rats were exposed (3 s) to a single inescapable electrical footshock (0.6 mA). Prior to the retention trial of a Y-maze task and the probe trials of two different learning strategy tasks, some of the rats that were exposed to the footshock (n = 17) were reminded of the stressor on an IA retrieval trial. Both groups of rats exposed to the initial stressor exhibited hypoactivity, but no impairment in spatial memory, on the Y-maze task conducted 1 week after exposure to the footshock. One month after exposure to footshock, both groups of rats exposed to the initial stressor tended to prefer a striatum-dependent learning strategy on a water T-maze task. However, 2 months after exposure to footshock, only shocked rats that were reminded of the stressor exhibited a preference for a striatum-dependent learning strategy on a visible-platform water maze task, which corresponded with lower levels of activity in an open field. The results indicate that reminders of a stressor perpetuate the deleterious effects of stress on affective and cognitive processes.

Decreased sexual motivation and heightened anxiety in male Long-Evans rats are correlated with the memory for a traumatic event.

Individuals suffering from posttraumatic stress disorder (PTSD) frequently report disturbances in sexual functioning in addition to alterations in their affective behaviors. Notably, maladaptive cognitions and dysfunctional behaviors are perpetuated by the emergence of the intrusive thoughts that characterize the disorder. In rats, reminders of a traumatic event designed to simulate intrusive thoughts are associated with impairments in affective, social, and sexual behaviors. The current study examined the relationship between the memory for a traumatic event and changes in sexual and affective behaviors in male Long-Evans rats (N = 36). The trauma featured a combination stressor consisting of simultaneous exposure to a footshock and the odor of soiled cat litter. Memory for the trauma was reactivated by re-exposures to the context of the trauma in the absence of stressors and confirmed by assessing the percentage of time spent freezing. Following the second and final reminder, traumatized males exhibited reduced sexual motivation and increased anxiety, signified by longer latencies to achieve their first mount on a post-stress test of sexual behavior, and longer latencies to begin feeding in a novel environment, respectively. Correlational analyses revealed that decreased sexual motivation and heightened anxiety were predicted by the memory for the trauma as indicated by the time spent freezing during the re-exposures. The findings from the current study have implications for understanding the relationship between stress and sexual functioning and indicate that the impairments in sexual behavior that often occur in individuals with PTSD may be impacted by their memory for the trauma.

Learning strategy is influenced by trait anxiety and early rearing conditions in prepubertal male, but not prepubertal female rats.

Rodents solve dual-solution tasks that require navigation to a goal by adopting either a hippocampus-dependent place strategy or a striatum-dependent stimulus-response strategy. A variety of factors, including biological sex and emotional status, influence the choice of learning strategy. In these experiments, we investigated the relationship between learning strategy and anxiety level in male and female rats prior to the onset of puberty, before the activational effects of gonadal hormones influence these processes. In the first experiment, prepubertal male rats categorized as high in trait anxiety at 26days of age exhibited a bias toward stimulus-response strategy at 28days of age, whereas age-matched females exhibited no preference in strategy regardless of anxiety level. In the second experiment, male and female rats were separated from their dams for either 15 or 180min per day during the first 2weeks of life and tested on a battery of anxiety and cognitive tasks between 25 and 29days of age. Prolonged maternal separations for 180min were associated with impaired spatial memory on a Y-maze task in both prepubertal males and females. Furthermore, prolonged maternal separations were linked to elevated anxiety and a bias for stimulus-response strategy in prepubertal males but not females. Alternatively, brief separations from dams for 15min were associated with intact spatial memory, lower levels of anxiety, and no preference for either learning strategy in both sexes. These results provide evidence of sex-specific effects of trait anxiety and early maternal separation on the choice of learning strategy used by prepubertal rodents.

The effects of biological sex and gonadal hormones on learning strategy in adult rats.

When learning to navigate toward a goal in a spatial environment, rodents employ distinct learning strategies that are governed by specific regions of the brain. In the early stages of learning, adult male rats prefer a hippocampus-dependent place strategy over a striatum-dependent response strategy. Alternatively, female rats exhibit a preference for a place strategy only when circulating levels of estradiol are elevated. Notably, male rodents typically perform better than females on a variety of spatial learning tasks, which are mediated by the hippocampus. However, limited research has been done to determine if the previously reported male spatial advantage corresponds with a greater reliance on a place strategy, and, if the male preference for a place strategy is impacted by removal of testicular hormones. A dual-solution water T-maze task, which can be solved by adopting either a place or a response strategy, was employed to determine the effects of biological sex and hormonal status on learning strategy. In the first experiment, male rats made more correct arm choices than female rats during training and exhibited a bias for a place strategy on a probe trial. The results of the second experiment indicated that testicular hormones modulated arm choice accuracy during training, but not the preference for a place strategy. Together, these findings suggest that the previously reported male spatial advantage is associated with a greater reliance on a place strategy, and that only performance during the training phase of a dual-solution learning task is impacted by removal of testicular hormones.

The relationships between trait anxiety, place recognition memory, and learning strategy.

Rodents learn to navigate mazes using various strategies that are governed by specific regions of the brain. The type of strategy used when learning to navigate a spatial environment is moderated by a number of factors including emotional states. Heightened anxiety states, induced by exposure to stressors or administration of anxiogenic agents, have been found to bias male rats toward the use of a striatum-based stimulus-response strategy rather than a hippocampus-based place strategy. However, no study has yet examined the relationship between natural anxiety levels, or trait anxiety, and the type of learning strategy used by rats on a dual-solution task. In the current experiment, levels of inherent anxiety were measured in an open field and compared to performance on two separate cognitive tasks, a Y-maze task that assessed place recognition memory, and a visible platform water maze task that assessed learning strategy. Results indicated that place recognition memory on the Y-maze correlated with the use of place learning strategy on the water maze. Furthermore, lower levels of trait anxiety correlated positively with better place recognition memory and with the preferred use of place learning strategy. Therefore, competency in place memory and bias in place strategy are linked to the levels of inherent anxiety in male rats.

Effects of early rearing conditions on cognitive performance in prepubescent male and female rats.

The interactions between a mother and her offspring during early postnatal life impact cognitive development in altricial species. The current study examined the influence of postnatal rearing conditions on subsequent cognitive functioning in male and female Long-Evans rats prior to puberty. Maternal conditions were manipulated by repeated separations of rat pups from their dams on postnatal days 2 though 14. In the early handling condition, pups were removed from mothers briefly for 15min daily, while in the maternal separation condition pups were separated from their mothers for 180min daily. Offspring from handled or separated litters were evaluated prior to puberty between days 25-36 of life on a battery of cognitive tasks that assessed several types of memory. Male rats separated from mothers for 180min were impaired in their non-spatial and spatial memory compared to early-handled males as indicated by their performance on an object recognition task, a Y-maze task, and reference and working memory versions of the water maze task. In contrast, maternally-separated females were not impaired, and in some cases performed better on memory tasks, compared to early-handled females. Results indicate that the biological sex of offspring moderated the effects of maternal conditions on diverse cognitive tasks. Because sex differences were evident prior to puberty, gonadal hormones likely had a limited influence on cognition. Although the bases for sex differences in the cognitive response to rearing conditions are unknown, disparities in maternal attentiveness directed toward male and female offspring may play a role.

Eye movement during REM sleep in children with attention deficit hyperactivity disorder.

The current study examined eye movements during rapid eye movement (REM) sleep of children ages 6-10 with attention deficit hyperactivity disorder (ADHD) and a control group without any known medical or psychiatric diagnoses. Electro-ocular recordings from archived polysomnograms were evaluated. An in-depth analysis revealed significantly lower frequency, higher amplitude eye movement in those with ADHD (N = 13) compared to the control group (N = 16). Although the results of this study are novel, defining distinct differences in eye movement during REM sleep of children with ADHD has the potential to supplement current biopsychosocial diagnostic models and further the understanding of the neurodevelopmental basis of ADHD.