Jill M. Daniel

Behavioral and Anatomical Correlates of Chronic Episodic Hypoxia during Sleep in the Rat

The role played by chronic episodic hypoxia (EHYP) in the neurocognitive morbidity of obstructive sleep apnea (OSA) is unknown. Sleep recordings, Morris water maze experiments, and immunohistochemistry for NMDA NR1 glutamate receptor, c-fos protein, and apoptosis [nuclear immunoreactivity for single-stranded DNA and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay] were conducted in EHYP-exposed Sprague Dawley male rats. Exposures consisted of up to14 d in an environmental chamber in which O2 concentrations were cycled between 10 and 21% every 90 sec or 30 min during 12 hr of daylight. For the remaining 12 hr, EHYP rats breathed room air, while controls spent 14 d in room air. Although EHYP induced significant disruption of sleep architecture during the initial day of exposure, sleep patterns normalized thereafter. Marked increases in apoptosis occurred in the CA1 hippocampal region (sevenfold) and cortex (Cx; eightfold) after 1–2 d of EHYP but not in CA3 and were followed by decreases toward normoxic levels by 14 d. Double labeling for NMDA NR1 and c-fos revealed marked architectural disorganization in CA1 and Cx with increases in c-fos over time. Rats exposed to EHYP displayed significantly longer escape latencies and swim path lengths to escape a hidden platform during 12 training trials given over 2 d. Differences in the performances of EHYP and control rats, although reduced, persisted after 14 d of recovery. We conclude that EHYP is associated with marked cellular changes over time within neural regions associated with cognitive functions. Furthermore, EHYP impaired performance during acquisition of a cognitive spatial task without affecting sensorimotor function. Such changes may underlie components of the learning and memory impairments found in OSA.

Estrogen enhances performance of female rats during acquisition of a radial arm maze

Estrogen can influence the expression of behaviors not associated directly with reproduction, including learning and memory. However, the effects of estrogen on learning and memory in mammals are complex, dependent on a variety of factors. The radial arm maze is a traditional experimental task that takes advantage of the natural foraging strategy of rats and provides an appropriate measure for studying the effects of estrogen on working memory in this species. In the experiments reported here, ovariectomized rats were implanted subcutaneously with 5-mm Silastic capsules containing 25% estradiol diluted with cholesterol. Control females received 5-mm Silastic capsules containing 100% cholesterol. Results of three separate experiments demonstrated that estradiol administered by Silastic implants for 30 days prior to eight-arm radial maze training, during the 24 days of maze training, or both significantly improved working memory performance compared to females treated with cholesterol alone, as indicated by improved arm choice accuracy over trials. The positive effect of estradiol exposure prior to training suggests that estrogen may induce neuronal changes that persist beyond the period of exposure with functional consequences for behavior.

Effects of Ovarian Hormones and Environment on Radial Maze and Water Maze Performance of Female Rats

The effects of gonadal hormones and environment on performance in an eight-arm radial maze and in the Morris water maze were determined in female rats. Long-Evans female rats were ovariectomized or sham ovariectomized at 35 days of age, and housed in complex environments or in isolation for the duration of the study. One month following surgeries, spatial working memory performance in the radial maze was assessed. Exposure to complex environmental conditions independently enhanced performance, as indicated by increased arm choice accuracy during 20 days of maze training. Additionally, gonadally intact females significantly outperformed ovariectomized females before cyclicity was disrupted by food deprivation. Following radial maze training, spatial reference memory performance was assessed in the same females utilizing the Morris water maze. Gonadally intact females housed in isolation performed significantly more poorly during 16 days of place training trials and displayed significantly shorter times in the platform quadrants and fewer target crossings during probe trials than gonadally intact and ovariectomized females housed in complex environments and ovariectomized rats housed in isolation. Consequently, acquisition and retention of the water maze was impaired by the presence of ovaries, and this impairment was counteracted by exposure to complex environments. Performance did not differ between groups on cued trials, indicating that sensorimotor and motivational functions did not differ between groups. Results of these experiments indicate that endogenous gonadal hormones can differentially affect performance on tasks of spatial working and spatial reference memory, and that environmental conditions can interact with gonadal hormones to affect behavior.

Estrogens, estrogen receptors, and female cognitive aging: The impact of timing

Estrogens have been shown to be protective agents against neurodegeneration and associated cognitive decline in aging females. However, clinical data have been equivocal as to the benefits to the brain and cognition of estrogen therapy in postmenopausal women. One factor that is proposed to be critical in determining the efficacy of hormone therapy is the timing of its initiation. The critical period or window of opportunity hypothesis proposes that following long-term ovarian hormone deprivation, the brain and cognition become insensitive to exogenously administered estrogens. In contrast, if estrogens are administered during a critical period near the time of cessation of ovarian function, they will exert beneficial effects. The focus of the current review is the examination of evidence from rodent models investigating the critical period hypothesis. A growing body of experimental data indicates that beneficial effects of 17β-estradiol (estradiol) on cognition and on cholinergic function and hippocampal plasticity, both of which have been linked to the ability of estradiol to exert beneficial effects on cognition, are attenuated if estradiol is administered following a period of long-term ovarian hormone deprivation. Further, emerging data implicate loss of estrogen receptor alpha (ERα) in the brain resulting from long-term hormone deprivation as a basis for the existence of the critical period. A unifying model is proposed by which the presence or absence of estrogens during a critical period following the cessation of ovarian function permanently alters the system resulting in decreased or increased risk, respectively, of neurodegeneration and cognitive decline.

Transient Estradiol Exposure during Middle Age in Ovariectomized Rats Exerts Lasting Effects on Cognitive Function and the Hippocampus

We determined whether transient exposure to estradiol during middle age in ovariectomized rats would exert lasting effects on cognition and the brain beyond the period of exposure. Two experiments were conducted. Rats 10-11 months of age were ovariectomized and received vehicle control treatment throughout the experiment, continuous estradiol treatment throughout the experiment, or 40 d of transient exposure to estradiol that ended 3 d before behavioral training. In the first experiment, rats were trained on a radial-maze working memory task and killed 2 months after the termination of transient exposure to estradiol. The hippocampus was immunostained for choline acetyltransferase and estrogen receptors alpha (ER alpha) and beta (ER beta) by Western blotting. In a second experiment to determine the durability of treatment effects, rats were behaviorally tested every other month until brains were collected for Western blotting 8 months after the termination of transient exposure to estradiol. Maze testing included delay trials and scopolamine trials, in which dose-effect curves for the muscarinic receptor antagonist were determined. Transient exposure to estradiol enhanced working memory and attenuated amnestic effects of scopolamine as effectively as continuous estradiol exposure. Enhancements persisted for up to 7 months. Transient exposure to estradiol increased hippocampal levels of ER alpha and choline acetyltransferase 2 months and ER alpha 8 months after termination of the exposure. Neither estradiol treatment altered estrogen receptor beta levels. Results demonstrate that short-term treatment with estradiol during middle age enhances working memory well beyond the duration of treatment and suggest ER alpha as a potential mechanism for this effect.

The critical period hypothesis of estrogen effects on cognition: Insights from basic research.

BACKGROUND In addition to its primary role in reproduction estrogen impacts brain areas important for cognition, including the hippocampus and prefrontal cortex. It has been hypothesized that decline in estrogen levels in women following menopause is associated with, or can exacerbate, age-related cognitive decline. However, clinical evidence to support a role for estrogen in preventing cognitive decline in women as they age is equivocal. The critical period hypothesis of estrogen effects on cognition, which proposes that estrogen administration has to be initiated within a critical time period following the loss of ovarian function in order for it to exert positive effects on the central nervous system, is offered as one explanation for inconsistencies across studies. SCOPE OF REVIEW This review details results from basic research using rodent models investigating the effects of estrogen on cognition in the aging female. Emphasis is placed on work investigating effects of timing of initiation of estrogen administration on its subsequent efficacy. MAJOR CONCLUSIONS Results of basic research provide support for the critical period hypothesis. Furthermore, results of work in rodent models suggest mechanisms by which the response to estrogen is altered if treatment is initiated following long-term ovarian hormone deprivation. GENERAL SIGNIFICANCE Understanding if and under what conditions hormone administration following the loss of ovarian function positively affects the brain and behavior could have important implications with regard to female cognitive aging. Results of basic research can contribute to this understanding and provide insight into the complex mechanisms by which estrogen affects cognition.

Long‐Term Ovarian Hormone Deprivation Alters the Ability of Subsequent Oestradiol Replacement to Regulate Choline Acetyltransferase Protein Levels in the Hippocampus and Prefrontal Cortex of Middle‐Aged Rats

The role of oestrogen replacement therapy in preventing or delaying age‐associated cognitive decline is controversial. Therapy success may critically depend on the time of treatment initiation following cessation of ovarian function. The present study aimed to assess, in middle‐aged rats, whether the ability of oestradiol to modulate the cholinergic system depends on the timing of treatment initiation following ovariectomy. Using western blotting, protein levels of choline acetyltransferase (ChAT) were measured in the hippocampus and prefrontal cortex (PFC), which are both important areas with respect to cognitive function. In an initial experiment, we established the effects of oestradiol delivered via implanted capsules on ChAT levels in the hippocampus and PFC of young adult animals. In a second experiment, we tested the ability of the same oestradiol treatment paradigm to affect ChAT protein in 15‐month‐old middle‐aged rats that had been ovariectomised either at the age of 10 months or at 15 months. In both experiments, rats were sacrificed 10 days after receiving implants and ChAT protein levels were measured. In both young adult and middle‐aged animals, oestradiol treatment initiated immediately after ovariectomy significantly increased ChAT levels in the hippocampus but not in the PFC compared to cholesterol control treatment. However, when oestradiol treatment was initiated 5 months after ovariectomy, it failed to significantly increase ChAT levels in the hippocampus, but did so in the PFC. These data indicate that, after prolonged ovarian hormone deprivation, the ability of subsequent oestradiol treatment to modulate ChAT protein levels is altered in a site‐specific manner.

The Ability of Oestradiol Administration to Regulate Protein Levels of Oestrogen Receptor Alpha in the Hippocampus and Prefrontal Cortex of Middle-Aged Rats is Altered Following Long-Term Ovarian Hormone Deprivation

Beneficial effects of oestrogen administration on cognition are attenuated if treatment is initiated following long‐term ovarian hormone deprivation. The mechanisms underlying this attenuation are unknown. The present study aimed to assess the effects of long‐term ovarian hormone deprivation on the ability of subsequent oestradiol treatment to regulate oestrogen receptor (ER) α and ERβ, and steroid receptor coactivator (SRC)‐1 in the hippocampus and prefrontal cortex of middle‐aged rats. In an initial experiment to assess oestradiol regulation of these proteins, 2‐month‐old rats were ovariectomised and immediately implanted with capsules containing cholesterol or oestradiol. Brains were collected 10 days later. In a second experiment, middle‐aged (10‐month‐old) rats were ovariectomised or underwent sham surgeries. Five months later, sham‐operated rats were ovariectomised and received oestradiol implants. Previously ovariectomised rats underwent sham surgeries and received oestradiol or cholesterol implants. Protein levels of ERα, ERβ, and SRC‐1 were measured following 10 days of oestradiol treatment using western blotting. In young animals, oestradiol treatment significantly increased ERα in the hippocampus and prefrontal cortex relative to control treatment. In middle‐aged animals, immediate oestradiol treatment significantly increased ERα in hippocampus, but not the prefrontal cortex. However, delayed oestradiol treatment failed to significantly increase ERα protein levels in hippocampus, but did so in prefrontal cortex. Levels of ERβ and SRC‐1 were unaffected by oestradiol treatment in either brain area in either of the age groups. These data indicate that prolonged ovarian hormone deprivation alters the ability of subsequent oestradiol replacement to regulate ERα protein levels in brain areas important for cognition.

The beneficial effects of estradiol on attentional processes are dependent on timing of treatment initiation following ovariectomy in middle-aged rats

The goal of the present study was to explore the effects of long-term hormone deprivation on the ability of subsequent estrogen treatment to affect attention performance on the 5-choice serial reaction time task (5-CSRTT). In an initial experiment to assess estradiol effects in young adults, 2-month-old rats were trained on the 5-CSRTT, then ovariectomized and immediately implanted with capsules containing cholesterol (n=10) or estradiol (n=10). Then rats were tested on the 5-CSRTT under baseline task parameters, under increased task difficulty (behavior challenge condition), and finally in muscarinic and nicotinic drug challenge conditions. In a second experiment, 10-month-old rats were trained on the 5-CSRTT and at 12 or 17 months of age rats were ovariectomized and treated with estradiol or cholesterol, so that one group received continuous cholesterol control treatment, two groups received estradiol treatment immediately following ovariectomy (either at 12 or 17 months), and one group received delayed estradiol treatment initiated 5 months following ovariectomies. At 17 months of age, rats were tested on the 5-CSRTT. Baseline performance was comparable between estradiol- and cholesterol-treated rats of both age groups. However, young estradiol-treated rats outperformed controls when behavior was challenged by shortening the intertrial interval (Short ITI). In the same Short ITI condition, middle-aged rats receiving immediate estradiol treatment beginning at the age of 17 months, but not 12 months, outperformed controls as well as animals receiving delayed estradiol treatment. No differences between groups were found in the cholinergic drug challenge conditions. These data indicate that chronic estradiol treatment for approximately 1 month but not 6 months is able to enhance attention performance, and that prolonged ovarian hormone deprivation attenuates these beneficial effects of subsequent estradiol treatment. These findings have implications for informing clinical research about the importance of timing and duration of hormone treatment.

Increased daily handling of ovariectomized rats enhances performance on a radial-maze task and obscures effects of estradiol replacement

Estrogen impacts performance on tasks of learning and memory, although there are inconsistencies in the direction and magnitude of the reported effects. Contributory factors to the inconsistencies may be methodological differences associated with different regimens of treatment. The goal of the present experiment was to assess the effect of increased handling, such as that commonly associated with pharmacological or other experimental manipulations, on the ability of estrogen to influence working memory performance. Young adult rats were ovariectomized and implanted with capsules containing either cholesterol or 25% estradiol diluted in cholesterol. Half of each hormone treatment group received standard handling, which consisted of handling required to carry out experimental procedures and half received increased handling, which consisted of standard handling as well as 2 min of additional daily handling by the experimenter. Animals were trained daily on a working memory task on an eight-arm radial maze for 24 days of acquisition and for eight additional daily trials in which delays of either 1 min or 3 h were imposed between the fourth and fifth arm choices. Animals that received increased handling exhibited significantly enhanced performance during acquisition and delay trials compared to those that received standard handling. Estradiol significantly enhanced performance during delay trials in animals that received standard handling but had no effect in animals that received increased handling. These results suggest that the amount of handling that animals receive as part of experimental procedures may obscure the memory enhancing effects of estradiol replacement on certain tasks of cognition.