Estrogen Receptor ß Contributes to Both Hypertension and Hypothalamic Plasticity in a Mouse Model of Peri-Menopause.

Hypertension susceptibility in women increases at the transition to menopause, termed perimenopause, a state characterized by erratic estrogen fluctuation and extended hormone cycles. Elucidating the role of estrogen signaling in the emergence of hypertension during perimenopause has been hindered by animal models that are confounded by abrupt estrogen cessation or effects of aging. In the present study, accelerated ovarian failure (AOF) in estrogen receptor ß (ERß) reporter mice was induced by 4-vinylcyclohexene diepoxide in young mice to model early-stage ovarian failure (peri-AOF) characteristic of peri-menopause. It was found that administering ERß agonists suppressed elevated blood pressure in a model of neurogenic hypertension induced by angiotensin II (AngII) in peri-AOF, but not in age-matched male mice. It was also found that ERß agonist administration in peri-AOF females, but not males, suppressed the heightened NMDAR signaling and reactive oxygen production in ERß neurons in the hypothalamic paraventricular nucleus (PVN), a critical neural regulator of blood pressure. It was further shown that deleting ERß in the PVN of gonadally intact females produced a phenotype marked by a sensitivity to AngII hypertension. These results suggest that ERß signaling in the PVN plays an important role in blood pressure regulation in female mice and contributes to hypertension susceptibility in females at an early stage of ovarian failure comparable to human perimenopause.

Extinction of Contextual Cocaine Memories Requires Cav1.2 within D1R-Expressing Cells and Recruits Hippocampal Cav1.2-Dependent Signaling Mechanisms.

Exposure to cocaine-associated contextual cues contributes significantly to relapse. Extinction of these contextual associations, which involves a new form of learning, reduces cocaine-seeking behavior; however, the molecular mechanisms underlying this process remain largely unknown. We report that extinction, but not acquisition, of cocaine conditioned place preference (CPP) in male mice increased Cav1.2 L-type Ca2+ channel mRNA and protein in postsynaptic density (PSD) fractions of the hippocampus, a brain region involved in drug-context associations. Moreover, viral-mediated deletion of Cav1.2 in the dorsal hippocampus attenuated extinction of cocaine CPP. Molecular studies examining downstream Cav1.2 targets revealed that extinction recruited calcium/calmodulin (Ca2+/CaMK)-dependent protein kinase II (CaMKII) to the hippocampal PSD. This occurred in parallel with an increase in phosphorylation of the AMPA GluA1 receptor subunit at serine 831 (S831), a CaMKII site, along with an increase in total PSD GluA1. The necessity of S831 GluA1 was further demonstrated by the lack of extinction in S831A GluA1 phosphomutant mice. Of note hippocampal GluA1 levels remained unaltered at the PSD, but were reduced near the PSD and at perisynaptic sites of dendritic spines in extinction-resistant S831A mutant mice. Finally, conditional knock-out of Cav1.2 in dopamine D1 receptor (D1R)-expressing cells resulted in attenuation of cocaine CPP extinction and lack of extinction-dependent changes in hippocampal PSD CaMKII expression and S831 GluA1 phosphorylation. In summary, we demonstrate an essential role for the hippocampal Cav1.2/CaMKII/S831 GluA1 pathway in cocaine CPP extinction, with data supporting contribution of hippocampal D1R-expressing cells in this process. These findings demonstrate a novel role for Cav1.2 channels in extinction of contextual cocaine-associated memories.SIGNIFICANCE STATEMENT Continued drug-seeking behavior, a defining characteristic of cocaine addiction, can be precipitated by contextual cues, yet the molecular mechanisms required for extinction of these context-specific memories remain poorly understood. Here, we have uncovered a novel and selective role of the Cav1.2 L-type Ca2+ channel and its downstream signaling pathway in the hippocampus that mediate extinction of cocaine conditioned place preference (CPP). We additionally provide evidence that supports a role of Cav1.2 within dopamine D1 receptor-expressing cells of the hippocampus for extinction of cocaine CPP. Therefore, these findings reveal a previously unknown role of Cav1.2 channels within the hippocampus and in D1 receptor-expressing cells in extinction of cocaine-associated memories, providing a framework for further exploration of mechanisms underlying extinction of cocaine-seeking behavior.

Redistribution of NMDA Receptors in Estrogen-Receptor-ß-Containing Paraventricular Hypothalamic Neurons following Slow-Pressor Angiotensin II Hypertension in Female Mice with Accelerated Ovarian Failure.

Hypertension in male and aging female rodents is associated with glutamate-dependent plasticity in the hypothalamus, but existing models have failed to capture distinct transitional menopausal phases that could have a significant impact on the synaptic plasticity and emergent hypertension. In rodents, accelerated ovarian failure (AOF) induced by systemic injection of 4-vinylcyclohexane diepoxide mimics the estrogen fluctuations seen in human menopause including the perimenopause transition (peri-AOF) and postmenopause (post-AOF). Thus, we used the mouse AOF model to determine the impact of slow-pressor angiotensin II (AngII) administration on blood pressure and on the subcellular distribution of obligatory N-methyl-D-aspartate (NMDA) receptor GluN1 subunits in the paraventricular hypothalamic nucleus (PVN), a key estrogen-responsive cardiovascular regulatory area. Estrogen-sensitive neuronal profiles were identified in mice expressing enhanced green fluorescent protein under the promoter for estrogen receptor (ER) ß, a major ER in the PVN. Slow-pressor AngII increased arterial blood pressure in mice at peri- and post-AOF time points. In control oil-injected (nonhypertensive) mice, AngII decreased the total number of GluN1 in ERß-containing PVN dendrites. In contrast, AngII resulted in a reapportionment of GluN1 from the cytoplasm to the plasma membrane of ERß-containing PVN dendrites in peri-AOF mice. Moreover, in post-AOF mice, AngII increased total GluN1, dendritic size and radical production in ERß-containing neurons. These results indicate that unique patterns of hypothalamic glutamate receptor plasticity and dendritic structure accompany the elevated blood pressure in peri- and post-AOF time points. Our findings suggest the possibility that distinct neurobiological processes are associated with the increased blood pressure during perimenopausal and postmenopausal periods.

NMDA Receptor Plasticity in the Hypothalamic Paraventricular Nucleus Contributes to the Elevated Blood Pressure Produced by Angiotensin II.

Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulation of the hypothalamic paraventricular nucleus (PVN). Many forms of glutamate-dependent plasticity are mediated by NMDA receptor GluN1 subunit expression and the distribution of functional receptor to the plasma membrane of dendrites. Here, we use a combined ultrastructural and functional analysis to examine the relationship between PVN NMDA receptors and the blood pressure increase induced by chronic infusion of a low dose of Ang II. We report that the increase in blood pressure produced by a 2 week administration of a subpressor dose of Ang II results in an elevation in plasma membrane GluN1 in dendrites of PVN neurons in adult male mice. The functional implications of these observations are further demonstrated by the finding that GluN1 deletion in PVN neurons attenuated the Ang II-induced increases in blood pressure. These results indicate that NMDA receptor plasticity in PVN neurons significantly contributes to the elevated blood pressure mediated by Ang II.

Female protection from slow-pressor effects of angiotensin II involves prevention of ROS production independent of NMDA receptor trafficking in hypothalamic neurons expressing angiotensin 1A receptors.

Renin­angiotensin system overactivity, upregulation of postsynaptic NMDA receptor function, and increased reactive oxygen species (ROS) production in the hypothalamic paraventricular nucleus (PVN) are hallmarks of angiotensin II (AngII)-induced hypertension, which is far more common in young males than in young females. We hypothesize that the sex differences in hypertension are related to differential AngII-induced changes in postsynaptic trafficking of the essential NMDA receptor GluN1 subunit and ROS production in PVN cells expressing angiotensin Type 1a receptor (AT1aR). We tested this hypothesis using slow-pressor (14-day) infusion of AngII (600 ng/kg/min) in mice, which elicits hypertension in males but not in young females. Two-month-old male and female transgenic mice expressing enhanced green fluorescent protein (EGFP) in AT1aR-containing cells were used. In males, but not in females, AngII increased blood pressure and ROS production in AT1aR­EGFP PVN cells at baseline and following NMDA treatment. Electron microscopy showed that AngII increased cytoplasmic and total GluN1­silver-intensified immunogold (SIG) densities and induced a trend toward an increase in near plasmalemmal GluN1­SIG density in AT1aR­EGFP dendrites of males and females. Moreover, AngII decreased dendritic area and diameter in males, but increased dendritic area of small (<1 µm) dendrites and decreased diameter of large (>1 µm) dendrites in females. Fluorescence microscopy revealed that AT1aR and estrogen receptor ß do not colocalize, suggesting that if estrogen is involved, its effect is indirect. These data suggest that the sexual dimorphism in AngII-induced hypertension is associated with sex differences in ROS production in AT1aR-containing PVN cells but not with postsynaptic NMDA receptor trafficking.

Sex and age differentially affect GABAergic neurons in the mouse prefrontal cortex and hippocampus following chronic intermittent hypoxia.

Obstructive sleep apnea (OSA), a chronic sleep disorder characterized by repetitive reduction or cessation of airflow during sleep, is widely prevalent and is associated with adverse neurocognitive sequelae including increased risk of Alzheimer's disease (AD). In humans, OSA is more common in elderly males. OSA is characterized by sleep fragmentation and chronic intermittent hypoxia (CIH), and recent epidemiological studies point to CIH as the best predictor of neurocognitive sequelae associated with OSA. The sex- and age- specific effects of OSA-associated CIH on specific cell populations such as γ-aminobutyric acid (GABA)-ergic neurons in the hippocampus and the medial prefrontal cortex (mPFC), regions important for cognitive function, remain largely unknown. The present study examined the effect of 35 days of either moderate (10% oxygen) or severe (5% oxygen) CIH on GABAergic neurons in the mPFC and hippocampus of young and aged male and female mice as well as post-accelerated ovarian failure (AOF) female mice. In the mPFC and hippocampus, the number of GABA-labeled neurons increased in aged and young severe CIH males compared to controls but not in young moderate CIH males. This change was not representative of the individual GABAergic cell subpopulations, as the number of parvalbumin-labeled neurons decreased while the number of somatostatin-labeled neurons increased in the hippocampus of severe CIH young males only. In all female groups, the number of GABA-labeled cells was not different between CIH and controls. However, in the mPFC, CIH increased the number of parvalbumin-labeled neurons in young females and the number of somatostatin-labeled cells in AOF females but decreased the number of somatostatin-labeled cells in aged females. In the hippocampus, CIH decreased the number of somatostatin-labeled neurons in young females. CIH decreased the density of vesicular GABA transporter in the mPFC of AOF females only. These findings suggest sex-specific changes in GABAergic neurons in the hippocampus and mPFC with males showing an increase of this cell population as compared to their female counterparts following CIH. Age at exposure and severity of CIH also differentially affect the GABAergic cell population in mice.

Olfactory association learning and brain-derived neurotrophic factor in an animal model of early deprivation.

Animal models can serve to explore neural mechanisms underlying the effects of stressful early experiences on behaviors supporting attachment. Neonatal rats primarily use olfaction for attachment, and Brain-Derived Neurotrophic Factor (BDNF) may be a key transcription target in olfactory association learning. In this experiment, neonatal male and female rats were isolated individually for 3 hr daily in the first week of life while their dams were left with partial litters (Early Deprivation, ED) or remained undisturbed (Control). At 1 week of age, subjects were tested using a 2-day classical conditioning paradigm. The conditioned group (O/M) was exposed to a novel odor paired with a milk infusion. Three additional groups included an unpaired odor and milk exposure group (O/M unP), an odor exposure alone group (O/NM), and neither an odor nor a milk group (NO/NM). Learning the odor association, as revealed in a position preference for the novel odor, was accompanied by an increase in hippocampal BDNF in O/M subjects from undisturbed Control litters. BDNF levels were also positively related to degree of preference for the odor in the O/M Control group. ED subjects did not make the classically conditioned odor association and did not show an increase in hippocampal BDNF. ED increased BDNF levels in the olfactory bulb compared to Controls regardless of training group; individual levels were not correlated with performance because samples were pooled. These results suggest that changes in the transcription of BDNF may underlie some of the long-term consequences of the early stress of maternal separation.

Plasma Membrane Affiliated AMPA GluA1 in Estrogen Receptor ß-containing Paraventricular Hypothalamic Neurons Increases Following Hypertension in a Mouse Model of Post-menopause.

Sex and ovarian function contribute to hypertension susceptibility, however, the mechanisms are not well understood. Prior studies show that estrogens and neurogenic factors, including hypothalamic glutamatergic NMDA receptor plasticity, play significant roles in rodent hypertension. Here, we investigated the role of sex and ovarian failure on AMPA receptor plasticity in estrogen-sensitive paraventricular nucleus (PVN) neurons in naïve and angiotensin II (AngII) infused male and female mice and female mice at early and late stages of accelerated ovarian failure (AOF). High-resolution electron microscopy was used to assess the subcellular distribution of AMPA GluA1 in age-matched male and female estrogen receptor beta (ERß) enhanced green fluorescent protein (EGFP) reporter mice as well as female ERß-EGFP mice treated with 4-vinylcyclohexene diepoxide. In the absence of AngII, female mice at a late stage of AOF displayed higher levels of GluA1 on the plasma membrane, indicative of functional protein, in ERß-expressing PVN dendrites when compared to male, naïve female and early stage AOF mice. Following slow-pressor AngII infusion, males, as well as early and late stage AOF females had elevated blood pressure. Significantly, only late stage-AOF female mice infused with AngII had an increase in GluA1 near the plasma membrane in dendrites of ERß-expressing PVN neurons. In contrast, prior studies reported that plasmalemmal NMDA GluN1 increased in ERß-expressing PVN dendrites in males and early, but not late stage AOF females. Together, these findings reveal that early and late stage AOF female mice display unique molecular signatures of long-lasting synaptic strength prior to, and following hypertension.

A laboratory exercise for a college-level, introductory neuroscience course demonstrating effects of housing environment on anxiety and psychostimulant sensitivity.

In this paper we describe a lab exercise developed for the Introduction to Neuroscience course at Williams College. One of a series of five labs, this exercise demonstrated several key principles of behavioral neuroscience. In this lab, students explored the effects of post-weaning housing environment on anxiety-like behavior and psychostimulant sensitivity in rodents. The exercise was intended to emphasize the importance and utility of animal models in neuroscience research and to give students hands-on experience with behavioral neuroscience research techniques. Students tested rats reared in social isolation or environmental enrichment for anxiety-like behaviors on the elevated plus maze, and for spontaneous and amphetamine-induced locomotor activity in the open field. They were then asked to analyze pooled class data and prepare a short lab report. Overall, student performance was excellent. This exercise emerged as a class favorite on course evaluations. Interestingly, the first time this exercise was conducted, the effects of environmental enrichment on anxiety-like behaviors and psychostimulant sensitivity were not consistent with those published in previous studies. Key methodological issues that may account for this discrepancy and contribute to successful implementation by other programs are discussed.

Alterations in the subcellular distribution of NADPH oxidase p47(phox) in hypothalamic paraventricular neurons following slow-pressor angiotensin II hypertension in female mice with accelerated ovarian failure.

At younger ages, women have a lower risk for hypertension than men, but this sexual dimorphism declines with the onset of menopause. These differences are paralleled in rodents following "slow-pressor" angiotensin II (AngII) administration: young male and aged female mice, but not young females, develop hypertension. There is also an established sexual dimorphism both in the cardiovascular response to the neurohypophyseal hormone arginine vasopressin (AVP) and in the expression of oxidative stress. We examined the relationship between AngII-mediated hypertension and the cellular distribution of the superoxide generating NADPH oxidase (NOX) in AVP-expressing hypothalamic paraventricular nucleus (PVN) neurons in "menopausal" female mice. Dual-labeling immunoelectron microscopy was used to determine whether the subcellular distribution of the organizer/adapter NOX p47(phox) subunit is altered in PVN dendrites following AngII administered (14 days) during the "postmenopausal" stage of accelerated ovarian failure (AOF) in young female mice treated with 4-vinylcyclohexene diepoxide. Slow-pressor AngII elevated blood pressure in AOF females and induced a significant increase in near plasmalemmal p47(phox) and a decrease in cytoplasmic p47(phox) in PVN AVP dendrites. These changes are the opposite of those observed in AngII-induced hypertensive male mice (Coleman et al. [2013] J. Neurosci. 33:4308-4316) and may be ascribed in part to baseline differences between young females and males in the near plasmalemmal p47(phox) on AVP dendrites seen in the present study. These findings highlight fundamental differences in the neural substrates of oxidative stress in the PVN associated with AngII hypertension in postmenopausal females compared with males. J. Comp. Neurol. 524:2251-2265, 2016. © 2015 Wiley Periodicals, Inc.

Sex differences in subcellular distribution of delta opioid receptors in the rat hippocampus in response to acute and chronic stress.

Drug addiction requires associative learning processes that critically involve hippocampal circuits, including the opioid system. We recently found that acute and chronic stress, important regulators of addictive processes, affect hippocampal opioid levels and mu opioid receptor trafficking in a sexually dimorphic manner. Here, we examined whether acute and chronic stress similarly alters the levels and trafficking of hippocampal delta opioid receptors (DORs). Immediately after acute immobilization stress (AIS) or one-day after chronic immobilization stress (CIS), the brains of adult female and male rats were perfusion-fixed with aldehydes. The CA3b region and the dentate hilus of the dorsal hippocampus were quantitatively analyzed by light microscopy using DOR immunoperoxidase or dual label electron microscopy for DOR using silver intensified immunogold particles (SIG) and GABA using immunoperoxidase. At baseline, females compared to males had more DORs near the plasmalemma of pyramidal cell dendrites and about 3 times more DOR-labeled CA3 dendritic spines contacted by mossy fibers. In AIS females, near-plasmalemmal DOR-SIGs decreased in GABAergic hilar dendrites. However, in AIS males, near-plasmalemmal DOR-SIGs increased in CA3 pyramidal cell and hilar GABAergic dendrites and the percentage of CA3 dendritic spines contacted by mossy fibers increased to about half that seen in unstressed females. Conversely, after CIS, near-plasmalemmal DOR-SIGs increased in hilar GABA-labeled dendrites of females whereas in males plasmalemmal DOR-SIGs decreased in CA3 pyramidal cell dendrites and near-plasmalemmal DOR-SIGs decreased hilar GABA-labeled dendrites. As CIS in females, but not males, redistributed DOR-SIGs near the plasmalemmal of hilar GABAergic dendrites, a subsequent experiment examined the acute affect of oxycodone on the redistribution of DOR-SIGs in a separate cohort of CIS females. Plasmalemmal DOR-SIGs were significantly elevated on hilar interneuron dendrites one-hour after oxycodone (3 mg/kg, I.P.) administration compared to saline administration in CIS females. These data indicate that DORs redistribute within CA3 pyramidal cells and dentate hilar GABAergic interneurons in a sexually dimorphic manner that would promote activation and drug related learning in males after AIS and in females after CIS.

Characterization of neural estrogen signaling and neurotrophic changes in the accelerated ovarian failure mouse model of menopause.

Accelerated ovarian failure (AOF) can be induced in young mice with low doses of 4-vinylcyclohexene diepoxide (VCD), modeling the hormone changes observed across menopause. We assessed markers of synaptic plasticity in the hippocampus, anxiety-like behavior, and spatial learning longitudinally at 4 time points across the AOF model: premenopause, early perimenopause, late perimenopause, and postmenopause (POST). As others have shown, VCD administration decreased ovarian follicle counts and increased acyclicity as the model progressed to POST but with no impact on organ or body weights. The morphology of Iba1 immunoreactive microglia did not differ between vehicle- and VCD-administered mice. Hippocampal postsynaptic density 95 levels were minimally altered across the AOF model but decreased at POST in CA3b 24 hours after exogenous estradiol benzoate (EB). In contrast, hippocampal phosphorylated AKT levels transiently decreased in premenopause but increased at POST after 24 hours of EB in select subregions. Electron microscopy revealed fewer estrogen receptor α containing dendritic spines and terminals in CA1 stratum radiatum at POST. mRNA levels of most brain-derived neurotrophic factor exons (except V and VI) were lower in POST compared with ovariectomized mice. Exon V was sensitive to 24 hours of EB administration in POST-VCD. Anxiety-like behavior was unaffected at any menopause phase. Spatial learning was unaffected in all groups, but POST-VCD mice performed below chance. Our results suggest that the AOF model is suitable for longitudinal studies of neurobiological changes across the menopause transition in mice. Our findings also point to complex interactions between estrogen receptors and pathways involved in synaptic plasticity.

Corticotropin-releasing factor in the mouse central nucleus of the amygdala: ultrastructural distribution in NMDA-NR1 receptor subunit expressing neurons as well as projection neurons to the bed nucleus of the stria terminalis.

Corticotropin-releasing factor (CRF) and glutamate are critical signaling molecules in the central nucleus of the amygdala (CeA). Central amygdala CRF, acting via the CRF type 1 receptor (CRF-R1), plays an integral role in stress responses and emotional learning, processes that are generally known to involve functional NMDA-type glutamate receptors. There is also evidence that CRF expressing CeA projection neurons to the bed nucleus of the stria terminalis (BNST) play an important role in stress related behaviors. Despite the potentially significant interactions between CRF and NMDA receptors in the CeA, the synaptic organization of these systems is largely unknown. Using dual labeling high resolution immunocytochemical electron microscopy, it was found that individual somata and dendrites displayed immunoreactivity for CRF and the NMDA-NR1 (NR1) subunit in the mouse CeA. In addition, CRF-containing axon terminals contacted postsynaptic targets in the CeA, some of which also expressed NR1. Neuronal profiles expressing the CRF type 1 receptor (CRF-R1), identified by the expression of green fluorescent protein (GFP) in bacterial artificial chromosome (BAC) transgenic mice, also contained NR1, and GFP immunoreactive terminals formed synapses with NR1 containing dendrites. Although CRF and GFP were only occasionally co-expressed in individual somata and dendritic profiles, contacts between labeled axon terminals and dendrites were frequently observed. A combination of tract tracing and immunocytochemistry revealed that a population of CeA CRF neurons projected to the BNST. It was also found that CRF, or GFP expressing terminals directly contacted CeA-BNST projection neurons. These results indicate that the NMDA receptor is positioned for the postsynaptic regulation of CRF expressing CeA neurons and the modulation of signals conveyed by CRF inputs. Interactions between CRF and NMDA receptor mediated signaling in CeA neurons, including those projecting to the BNST, may provide the synaptic basis for integrating the experience of stress and relevant environmental stimuli with behaviors that may be of particular relevance to stress-related learning and the emergence of psychiatric disorders, including drug addiction.

Sex and estrogen receptor expression influence opioid peptide levels in the mouse hippocampal mossy fiber pathway.

The opioid peptides, dynorphin (DYN) and enkephalin (L-ENK) are contained in the hippocampal mossy fiber pathway where they modulate synaptic plasticity. In rats, the levels of DYN and L-ENK immunoreactivity (-ir) are increased when estrogen levels are elevated (Torres-Reveron et al., 2008, 2009). Here, we used quantitative immunocytochemistry to examine whether opioid levels are similarly regulated in wildtype (WT) mice over the estrous cycle, and how these compared to males. Moreover, using estrogen receptor (ER) alpha and beta knock-out mice (AERKO and BERKO, respectively), the present study examined the role of ERs in rapid, membrane-initiated (6 h), or slower, nucleus-initiated (48 h) estradiol effects on mossy fiber opioid levels. Unlike rats, the levels of DYN and L-ENK-ir did not change over the estrous cycle. However, compared to males, females had higher levels of DYN-ir in CA3a and L-ENK-ir in CA3b. In WT and BERKO ovariectomized (OVX) mice, neither DYN- nor L-ENK-ir changed following 6 or 48 h estradiol benzoate (EB) administration. However, DYN-ir significantly increased 48 h after EB in the dentate gyrus (DG) and CA3b of AERKO mice only. These findings suggest that cyclic hormone levels regulate neither DYN nor L-ENK levels in the mouse mossy fiber pathway as they do in the rat. This may be due to species-specific differences in the mossy fiber pathway. However, in the mouse, DYN levels are regulated by exogenous EB in the absence of ERα possibly via an ERß-mediated pathway requiring new gene transcription.

Accelerated ovarian failure: a novel, chemically induced animal model of menopause.

Current rodent models of menopause fail to adequately recapitulate the menopause transition. The intact aging model fails to achieve very low estrogen levels, and the ovariectomy model lacks a perimenopause phase. A new rodent model of accelerated ovarian failure (AOF) successfully replicates human perimenopause and postmenopause, including estrous acyclicity and fluctuating, followed by undetectable, estrogen levels, and allows for the dissociation of the effects of hormone levels from the effects of aging. In this model, an ovotoxic chemical, 4-vinylcyclohexene diepoxide (VCD), selective for primary and primordial follicles, is injected intraperitonelly in animals for 15 days. As the mature follicle population is depleted through natural cycling, ovarian failure follows increasing periods of acyclity. Administered at low doses, VCD specifically causes apoptotic cell death of primordial follicles but does not affect other peripheral tissues, including the liver and spleen, nor does it affect brain inflammation markers. In addition to reducing confounds associated with genetic and surgical manipulations, the AOF model maintains the presence of ovarian tissue which importantly parallels to the menopause transition in humans. The VCD injection procedure can be applied to studies using transgenic or knockout mice strains, or in other disease-state models (e.g., ischemia, atherosclerosis, or diabetes). This AOF model of menopause will generate new insights into women's health particularly in determining the critical periods (i.e., a window of opportunity) during perimenopause for restoring ovarian hormones for the most efficacious effect on memory and mood disorders as well as other menopausal symptoms.