Robert B. Gibbs

Long-term treatment with estrogen and progesterone enhances acquisition of a spatial memory task by ovariectomized aged rats.

Female Sprague-Dawley rats were ovariectomized at 13 months of age. Four groups received different regimens of estrogen or estrogen plus progesterone replacement beginning either immediately, 3 months, or 10 months after ovariectomy and were compared with non-hormone-treated controls. Eight to twelve months after ovariectomy, animals were trained on a delayed matching-to-position (DMP) spatial memory task. Long-term treatment with estrogen or estrogen plus progesterone significantly enhanced acquisition of the DMP task by aged animals after long-term loss of ovarian function. Weekly administration of estrogen and progesterone was at least as effective as, if not more effective than, continuous treatment with estrogen alone. In addition, treatment initiated 3 months, but not 10 months, after ovariectomy was as effective at enhancing DMP acquisition as continuous estrogen treatment initiated immediately after ovariectomy, suggesting a window of opportunity after the loss of ovarian function during which hormone replacement can effectively prevent the effects of aging and hormone deprivation on cognitive function. These findings suggest that repeated treatment with estrogen and progesterone initiated within a specific period of time after the loss of ovarian function may be effective at preventing specific negative effects of hormone deprivation on brain aging and cognitive decline.

Levels of trkA and BDNF mRNA, but not NGF mRNA, fluctuate across the estrous cycle and increase in response to acute hormone replacement

Abstract Recent studies suggest that hormone replacement therapy can help to reduce the risk and severity of Alzheimers-related dementia in postmenopausal women. We have hypothesized that these effects are due, in part, to the ability for estrogen and progesterone to enhance hippocampal function, as well as the functional status of cholinergic projections to the hippocampus and cortex, by influencing the expression of specific neurotrophins and neurotrophin receptors. In the present study, quantitative in situ hybridization techniques were used to determine whether the levels of trkA mRNA in the basal forebrain, and nerve growth factor (NGF) mRNA and brain-derived neurotrophic factor (BDNF) mRNA in the hippocampus, are significantly affected by physiological changes in circulating gonadal steroids. Gonadally intact animals were sacrificed at different stages of the estrous cycle and ovariectomized animals were sacrificed at different times following the administration of either estrogen or estrogen plus progesterone. In gonadally intact animals, significant fluctuations in the levels of trkA mRNA in the medial septum (MS), and BDNF mRNA in regions CA1 and CA3/4 of the hippocampus, were detected across the estrous cycle. In animals that received hormone replacement, a significant increase (30.4%) in trkA mRNA was detected in the MS of animals sacrificed 24 h following estrogen administration. Levels of trkA mRNA in the MS declined to control levels over the next 48 h; however, a single injection of progesterone administered 48 h after estradiol appeared to prevent any further decline in trkA mRNA over the next 24 h. In addition, significant increases in BDNF mRNA were detected in the dentate granule cell layer (73.4%), region CA1 (28.1%), and region CA3/4 (76.9%) of animals sacrificed 53 h after receiving estrogen and 5 h after receiving progesterone. No significant changes in trkA mRNA were detected in the nucleus basalis magnocellularis, and no significant changes in NGF mRNA were detected in the hippocampus. These data demonstrate that levels of trkA mRNA in the MS, and BDNF mRNA in the hippocampus, are affected by physiological changes in the levels of circulating gonadal steroids and are elevated in response to acute hormone replacement. The relevance of these effects to the ability for estrogen replacement to enhance cholinergic activity and hippocampal function, and thereby reduce the risk and severity of Alzheimers-related dementia in postmenopausal women, is discussed.

Effects of Estrogen Replacement on the Relative Levels of Choline Acetyltransferase, trkA, and Nerve Growth Factor Messenger RNAs in the Basal Forebrain and Hippocampal Formation of Adult Rats

Previous studies have shown that estrogen replacement can significantly affect the expression of choline acetyltransferase immunoreactivity (ChAT-IR) and low-affinity (p75NGFR) nerve growth factor receptors within cholinergic neurons located in specific regions of the basal forebrain. To extend this work, we have examined the effects of estrogen replacement on relative levels of choline acetyltransferase (ChAT), trkA, and nerve growth factor (NGF) mRNAs within different regions of the basal forebrain and hippocampal formation using quantitative in situ hybridization techniques. Ovariectomized Sprague-Dawley rats received continuous estrogen replacement for 2 days, 1 week, or 2 weeks. The data show that estrogen replacement results in significant increases in relative cellular levels of ChAT mRNA in the medial septum (MS) and nucleus basalis magnocellularis (nBM), but not in the horizontal limb of the diagonal band of Broca (HDB) or the striatum. In contrast, estrogen replacement resulted in significant decreases in relative levels of NGF mRNA in the hippocampus and of trkA mRNA in the MS and nBM (but not in the HDB or the striatum). The time-course of these effects is consistent with estrogen having a direct effect on ChAT expression which is followed by indirect effects on trkA. The data are also consistent with previous findings in which estrogen replacement resulted in significant increases in ChAT-IR which were followed by significant decreases in p75NGFR mRNA and protein and then a reduction in ChAT-IR back to control levels. Together, these data indicate that estrogen replacement can have significant effects on basal forebrain cholinergic function, and that some of these effects may be mediated by effects of estrogen replacement on the expression of NGF and NGF receptors.

Sexual stimulation activates c-fos within estrogen-concentrating regions of the female rat forebrain.

Regions of the brain that concentrate estrogen and progesterone are thought to regulate female sexual behavior by altering gene expression and neural sensitivity to afferent stimulation. We used immunocytochemistry and in situ hybridization to examine c-fos gene expression within estrogen-concentrating regions of the forebrain following various types of sexual stimulation with or without hormone treatment. Ovariectomized rats received injections of estradiol benzoate 48 h and progesterone 4 h before testing. Control rats that had been ovariectomized at least 5 months before testing did not receive hormone treatment. Rats were then either placed into bilevel testing chambers with sexually vigorous males, received manual stimulation of the flanks, received vaginocervical stimulation with a glass rod, or were left in their home cages. Copulation with intromission and ejaculation in hormone-treated rats, or stimulation of the vaginal cervix in both hormone-treated and control rats, produced a dramatic induction of c-fos mRNA and Fos-like immunoreactivity in estrogen-concentrating regions, such as the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus, ventromedial hypothalamus, lateral habenula, and medial amygdala, in addition to regions that do not readily concentrate estrogen, such as the neocortex, thalamus, and striatum. Mechanical stimulation of the flanks produced a smaller induction of Fos in these rats, whereas hormone treatment alone had no effect. These data demonstrate that afferent sensory stimulation, but not estrogen or progesterone, regulates c-fos gene expression within different estrogen-concentrating and non-concentrating regions of the female rat forebrain.

Structural Interactions between Kisspeptin and GnRH Neurons in the Mediobasal Hypothalamus of the Male Rhesus Monkey (Macaca mulatta) as Revealed by Double Immunofluorescence and Confocal Microscopy

Kisspeptin is recognized to play a critical role in eliciting the pubertal resurgence of pulsatile GnRH release, the proximal trigger of puberty in higher primates. Expression of the kisspeptin receptor (GPR54) by GnRH neurons indicates a direct action of kisspeptin on the GnRH neuronal network. The purpose of the present study was to examine the distribution of kisspeptin cell bodies in the monkey hypothalamus and to assess the structural basis for the stimulatory action of kisspeptin on the GnRH neuronal network. Three castrated male rhesus monkeys, 39-51 months of age, were deeply anesthetized and their brains perfused transcardially with 4% paraformaldehyde in PBS. Serial 25-microm coronal sections throughout the hypothalamus were prepared, and immunopositive neurons identified using a cocktail of specific primary antibodies (sheep anti-kisspeptin at 1:120,000, and rabbit anti-GnRH at 1:100,000) detected with fluorescently tagged secondary antibodies (antisheep, Alexa Fluor 488; antirabbit, Cy3) in combination with confocal microscopy. Kisspeptin perikarya were found only in the mediobasal hypothalamus (MBH) almost exclusively in the posterior two-thirds of the arcuate nucleus. Surprisingly, kisspeptin-beaded axons made only infrequent contacts with GnRH neurons (kisspeptin and GnRH profiles abutting in a 0.5- to 1.0-mum optical section) in the MBH. In the median eminence, kisspeptin and GnRH axons were found in extensive and intimate association. GnRH contacts on kisspeptin perikarya and dendrites were observed. These findings indicate that nonsynaptic pathways of communication in the median eminence should be considered as a possible mechanism of kisspeptin regulation of GnRH release, and provide an anatomical basis for reciprocal control of kisspeptin neuronal activity by GnRH.

Estrogen and Basal Forebrain Cholinergic Neurons: Implications for Brain Aging and Alzheimer's Disease-Related Cognitive Decline☆

Recent studies suggest that estrogen replacement therapy can reduce the risk and severity of Alzheimers disease (AD)-related dementia in postmenopausal women. Many different mechanisms by which estrogen therapy may help to reduce the risk and severity of AD-related pathophysiology have been proposed. Recent animal studies suggest that one way in which estrogen replacement may help to reduce cognitive deficits associated with aging and AD is by enhancing the functional status of cholinergic projections to the hippocampus and cortex. Here we review the evidence that estrogen is important in the maintenance of cholinergic neurons projecting to the hippocampus and cortex and that estrogen replacement can enhance the functional status of these neurons, as well as reduce cognitive deficits associated with muscarinic cholinergic impairment. Based on these studies, we conclude that, in animals, short-term treatment with physiological levels of estrogen, or estrogen and progesterone, has significant positive effects on cholinergic neurons in the medial septum and nucleus basalis magnocellularis and on their projections to the hippocampus and cortex. We hypothesize that similar effects in humans may help delay the decline in basal forebrain cholinergic function associated with aging and AD and thereby reduce the risk and severity of AD-related dementia in postmenopausal women.

Effects of estrogen on basal forebrain cholinergic neurons vary as a function of dose and duration of treatment

Studies suggest that estrogen replacement can influence learning and memory processes via effects on cholinergic neurons located in specific regions of the basal forebrain. In the present study, immunocytochemical techniques were used to examine the effects of estrogen on basal forebrain cholinergic neurons as a function of the dose and duration of estrogen treatment. Ovariectomized rats received 2, 10, 25, or 100 microg estradiol every other day for a period of 1, 2, or 4 weeks. Sections through the basal forebrain were then processed for the detection of choline acetyltransferase (ChAT) or the low-affinity nerve growth factor receptor (p75NGFR), and the number of immunoreactive cells in the medial septum (MS), the horizontal limb of the diagonal band of Broca (HDB) and the nucleus basalis magnocellularis (NBM) were counted. The effects of dose and duration of estrogen treatment were evaluated by analysis of variance and individual group means were compared with ovariectomized controls using a two-tailed Dunnets test. Administration of 2, 10, or 25 microg estradiol for 1 week produced a dose-related increase in the number of ChAT-like immunoreactive (IR) cells detected in the MS. Likewise treatment with 10 microg estradiol for 1 week, or with 2 microg estradiol for 2 weeks resulted in a significant increase in the number of ChAT-IR cells detected in the NBM. These effects were not observed following treatment with higher doses of estradiol. Nor were they maintained following repeated administration of estradiol for longer periods of time. In contrast, repeated administration of estradiol for 2 or 4 weeks resulted in significant decreases in the number of p75NGFR-IR cells detected in the MS, with the greatest effects observed following treatment with the higher doses of estradiol for longer periods of time. These findings demonstrate that (1) estrogen replacement produces regionally selective effects on basal forebrain cholinergic neurons which vary as a function of both the dose and duration of estrogen treatment, and (2) estrogen has both short-term and longer-term effects on basal forebrain cholinergic neurons, each of which may contribute to the effects of estrogen on learning and memory process and the development of age- and disease-related cognitive decline.

Treatment with estrogen and progesterone affects relative levels of brain-derived neurotrophic factor mRNA and protein in different regions of the adult rat brain

Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine the effects of acute estrogen and progesterone replacement on relative levels of brain-derived neurotrophic factor (BDNF) mRNA and protein in different regions of the adult rat brain. Adult ovariectomized animals were killed 53 h after receiving estrogen (E53), 53 h after receiving estrogen and 5 h after receiving progesterone (E53P), or 72 h after receiving estrogen and 24 h after receiving progesterone (E72P). Ovariectomized controls were killed 53 and 72 h after receiving vehicle. Tissues from the hippocampus, pyriform cortex, olfactory bulbs, septum, and nucleus basalis/ventral pallidum were dissected. Tissues from the right hemisphere were processed for quantitative RT-PCR analysis of BDNF mRNA, and tissues from the left hemisphere were processed for the detection and quantification of BDNF protein by ELISA. The results demonstrate significant increases in BDNF mRNA in the pyriform cortex of E53- and E53P-treated animals, as well as an increase in BDNF protein in the pyriform cortex of E72P-treated animals, relative to controls. Significant increases in BDNF mRNA were likewise detected in the hippocampus of E53- and E72P-treated animals, but were accompanied by a significant decrease in BDNF protein in the hippocampus of E53P- and E72P-treated animals relative to controls. No significant changes in BDNF mRNA or protein were detected in the olfactory bulbs, frontal cortex, or nucleus basalis/ventral pallidum following hormone treatment; however, an increase in BDNF protein was detected in the septum of E53-treated animals. This may indicate an increase in the retrograde transport of BDNF from the hippocampus to the septum, which could help account for the decrease in BDNF protein detected in the hippocampus following hormone treatment. These findings demonstrate that hormone replacement significantly affects relative levels of BDNF mRNA and protein within specific regions of the brain. These effects may, in turn, contribute to the effects of estrogen replacement on hippocampal connectivity and cognitive processes that have recently been reported.

Effects of gonadal hormone replacement on measures of basal forebrain cholinergic function

The effects of different hormone replacement regimens on basal forebrain cholinergic function were examined by measuring changes in choline acetyltransferase activity and high affinity choline uptake in adult, ovariectomized, rats. Increases in choline acetyltransferase activity were detected in the frontal cortex (20. 1%) and olfactory bulbs (30.4%) following two weeks, but not four weeks, of repeated treatment with estrogen plus progesterone. Increases in high affinity choline uptake were detected in the frontal cortex (39.5-55.1%), hippocampus (34.9-48.9%), and olfactory bulbs (29.9%) after two weeks, but not four weeks, of either continuous estrogen administration, repeated progesterone administration, or repeated treatment with estrogen plus progesterone. Repeated administration of estradiol (2-25 microg/250 g body weight) for two or four weeks, and continuous estrogen administration for four weeks and six months, produced no significant changes in choline acetyltransferase activity or high affinity choline uptake in the hippocampus, frontal cortex or olfactory bulbs. Continuous estrogen administration for 13 months produced a significant decrease in high affinity choline uptake across all regions with the largest effect (-28.1%) detected in the hippocampus. The findings demonstrate that short-term treatment with estrogen and/or progesterone can significantly enhance cholinergic function within specific targets of the basal forebrain cholinergic projections. Most important is the fact that the effects varied considerably according to the manner and regimen of hormone replacement and did not persist with prolonged treatment. These findings could have important implications for the effective use of hormone replacement strategies in the prevention and treatment of Alzheimers disease and age-related cognitive decline in women.

Effects of estrogen and fimbria/fornix transection on p75NGFR and ChAT expression in the medial septum and diagonal band of Broca.

NGF receptor-expressing cells located in the basal forebrain have recently been shown to contain estrogen (E) receptors (Toran-Allerand and MacLusky. 1989. Soc. Neurosci. Abstr. 15: 954). In the present study, we have examined the effects of E-treatment on p75NGFR and choline acetyltransferase (ChAT) expression by neurons in the medial septum (MS) and the vertical (VDB) and horizontal (HDB) limbs of the diagonal band of Broca using immunocytochemical and in situ hybridization techniques. First, since E-treatment has been shown to affect neuronal survival and to stimulate synaptic reorganization and growth within various regions of the brain, we hypothesized that E-treatment might attenuate the loss of p75NGFR immunoreactivity (IR) which occurs in the MS and VDB following transection of the fimbria/fornix. Contrary to our hypothesis, E-treatment did not attenuate the effects of fimbria/fornix transection. In fact, E-treatment alone produced a significant decrease in the number of p75NGFR-IR cells detected in the MS. Subsequent experiments confirmed that chronic E-treatment produces a down-regulation of both p75NGFR-IR and p75NGFR mRNA in the MS and VDB. In the MS, estrogen appeared to affect a subpopulation of p75NGFR-expressing neurons which were also affected by fimbria/fornix transection since the effects of these two treatments were not additive. In addition, effects of E-treatment on p75NGFR-IR were sex-specific (observed in females but not in males) and were reversible in the MS after 2 weeks, but not after 4 weeks (allowing 2 weeks recovery), of E-treatment. A time-course analysis revealed that effects of E-treatment on p75NGFR-IR were not observed until after 16 days (MS) or 30 days (VDB) of E-treatment and were preceded by a significant and transient increase in ChAT expression in both the MS and VDB. The data are consistent with the possibility that continuous, long-term exposure to gonadal steroids may contribute to a loss of p75NGFR-expressing neurons with age. In addition, the data suggest that p75NGFR expression may play a role in regulating the functioning of specific basal forebrain cholinergic neurons. Different mechanisms by which E-treatment might influence ChAT and p75NGFR expression in brain are discussed.