Tracey A. Van Kempen

A Genetic Variant BDNF Polymorphism Alters Extinction Learning in Both Mouse and Human

Of Mice and Men Just how closely must mouse models replicate the known features of human disorders to be accepted as useful for mechanistic and therapeutic studies? Soliman et al. (p. 863, published online 14 January) compared mice that vary only in their allelic composition at one position within the gene encoding brain-derived neurotrophic factor (BDNF) with humans exhibiting the same range of allelic variation. Individuals (mice and humans) carrying the allele that codes for a methionine-containing variant of BDNF retained a fearful response to a threatening stimulus even after its removal in comparison to those with the valine variant. Furthermore, in both cases, this linkage was mediated by diminished activity in the ventral-medial region of the prefrontal cortex. This deficit in extinction learning may contribute to differential responses to extinction-based therapies for anxiety disorders. A common genetic variation affecting fear learning and extinction operates through the same pathways in mice and men. Mouse models are useful for studying genes involved in behavior, but whether they are relevant to human behavior is unclear. Here, we identified parallel phenotypes in mice and humans resulting from a common single-nucleotide polymorphism in the brain-derived neurotrophic factor (BDNF) gene, which is involved in anxiety-related behavior. An inbred genetic knock-in mouse strain expressing the variant BDNF recapitulated the phenotypic effects of the human polymorphism. Both were impaired in extinguishing a conditioned fear response, which was paralleled by atypical frontoamygdala activity in humans. Thus, this variant BDNF allele may play a role in anxiety disorders showing impaired learning of cues that signal safety versus threat and in the efficacy of treatments that rely on extinction mechanisms, such as exposure therapy.

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 womens 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.

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.

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.

Slow-pressor angiotensin II hypertension and concomitant dendritic NMDA receptor trafficking in estrogen receptor β-containing neurons of the mouse hypothalamic paraventricular nucleus are sex and age dependent.

The incidence of hypertension increases after menopause. Similar to humans, “slow‐pressor” doses of angiotensin II (AngII) increase blood pressure in young males, but not in young female mice. However, AngII increases blood pressure in aged female mice, paralleling reproductive hormonal changes. These changes could influence receptor trafficking in central cardiovascular circuits and contribute to hypertension. Increased postsynaptic N‐methyl‐D‐aspartate (NMDA) receptor activity in the hypothalamic paraventricular nucleus (PVN) is crucial for the sympathoexcitation driving AngII hypertension. Estrogen receptors β (ERβs) are present in PVN neurons. We tested the hypothesis that changes in ovarian hormones with age promote susceptibility to AngII hypertension, and influence NMDA receptor NR1 subunit trafficking in ERβ‐containing PVN neurons. Transgenic mice expressing enhanced green fluorescent protein (EGFP) in ERβ‐containing cells were implanted with osmotic minipumps delivering AngII (600 ng/kg/min) or saline for 2 weeks. AngII increased blood pressure in 2‐month‐old males and 18‐month‐old females, but not in 2‐month‐old females. By electron microscopy, NR1‐silver–intensified immunogold (SIG) was mainly in ERβ‐EGFP dendrites. At baseline, NR1‐SIG density was greater in 2‐month‐old females than in 2‐month‐old males or 18‐month‐old females. After AngII infusion, NR1‐SIG density was decreased in 2‐month‐old females, but increased in 2‐month‐old males and 18‐month‐old females. These findings suggest that, in young female mice, NR1 density is decreased in ERβ‐PVN dendrites thus reducing NMDA receptor activity and preventing hypertension. Conversely, in young males and aged females, NR1 density is upregulated in ERβ‐PVN dendrites and ultimately leads to the neurohumoral dysfunction driving hypertension. J. Comp. Neurol. 522:3075–3090, 2014.

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.

Renin–angiotensin 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 AT1aR–EGFP PVN cells at baseline and following NMDA treatment. Electron microscopy showed that AngII increased cytoplasmic and total GluN1–silver‐intensified immunogold (SIG) densities and induced a trend toward an increase in near plasmalemmal GluN1–SIG density in AT1aR–EGFP 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 β 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. Synapse 69:148–165, 2015.  © 2015 Wiley Periodicals, Inc.

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 ERβ-mediated pathway requiring new gene transcription.

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.

Redistribution of NMDA Receptors in Estrogen-Receptor-β-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) β, 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 ERβ-containing PVN dendrites. In contrast, AngII resulted in a reapportionment of GluN1 from the cytoplasm to the plasma membrane of ERβ-containing PVN dendrites in peri-AOF mice. Moreover, in post-AOF mice, AngII increased total GluN1, dendritic size and radical production in ERβ-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.

Alterations in the subcellular distribution of NADPH oxidase p47phox 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 p47phox 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 p47phox and a decrease in cytoplasmic p47phox 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 p47phox 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.