Estrous Cycle Mediates Midbrain Neuron Excitability Altering Social Behavior upon Stress.

The estrous cycle is a potent modulator of neuron physiology. In rodents, in vivo ventral tegmental area (VTA) dopamine (DA) activity has been shown to fluctuate across the estrous cycle. Although the behavioral effect of fluctuating sex steroids on the reward circuit is well studied in response to drugs of abuse, few studies have focused on the molecular adaptations in the context of stress and motivated social behaviors. We hypothesized that estradiol fluctuations across the estrous cycle acts on the dopaminergic activity of the VTA to alter excitability and stress response. We used whole-cell slice electrophysiology of VTA DA neurons in naturally cycling, adult female C57BL/6J mice to characterize the effects of the estrous cycle and the role of 17ß-estradiol on neuronal activity. We show that the estrous phase alters the effect of 17ß-estradiol on excitability in the VTA. Behaviorally, the estrous phase during a series of acute variable social stressors modulates subsequent reward-related behaviors. Pharmacological inhibition of estrogen receptors in the VTA before stress during diestrus mimics the stress susceptibility found during estrus, whereas increased potassium channel activity in the VTA before stress reverses stress susceptibility found during estrus as assessed by social interaction behavior. This study identifies one possible potassium channel mechanism underlying the increased DA activity during estrus and reveals estrogen-dependent changes in neuronal function. Our findings demonstrate that the estrous cycle and estrogen signaling changes the physiology of DA neurons resulting in behavioral differences when the reward circuit is challenged with stress.SIGNIFICANCE STATEMENT The activity of the ventral tegmental area encodes signals of stress and reward. Dopaminergic activity has been found to be regulated by both local synaptic inputs as well as inputs from other brain regions. Here, we provide evidence that cycling sex steroids also play a role in modulating stress sensitivity of dopaminergic reward behavior. Specifically, we reveal a correlation of ionic activity with estrous phase, which influences the behavioral response to stress. These findings shed new light on how estrous cycle may influence dopaminergic activity primarily during times of stress perturbation.

Angiotensin II differentially affects hippocampal glial inflammatory markers in young adult male and female mice.

Hypertension is a risk factor for neurodegenerative disorders involving inflammation and inflammatory cytokine-producing brain cells (microglia and astrocytes) in the hippocampus and medial prefrontal cortex (mPFC). Here we investigated the effect of slow-pressor angiotensin II (AngII) on gliosis in the hippocampus and mPFC of young adult (2-mo-old) male and female mice. In males, AngII induced hypertension, and this resulted in an increase in the density of the astrocyte marker glial fibrillary acidic protein (GFAP) in the subgranular hilus and a decrease in the density of the microglial marker ionized calcium binding adapter molecule (Iba-1) in the CA1 region. Females infused with AngII did not show hypertension but, significantly, showed alterations in hippocampal glial activation. Compared with vehicle, AngII-infused female mice had an increased density of Iba-1 in the dentate gyrus and CA2/3a region. Like males, females infused with AngII exhibited decreased Iba-1 in the CA1 region. Neither male nor female mice showed differences in GFAP or Iba-1 in the mPFC following AngII infusion. These results demonstrate that the hippocampus is particularly vulnerable to AngII in young adulthood. Differences in gonadal hormones or the sensitivity to AngII hypertension may account for divergences in GFAP and Iba-1 in males and females.

Tumor Necrosis Factor α Receptor Type 1 Activation in the Hypothalamic Paraventricular Nucleus Contributes to Glutamate Signaling and Angiotensin II-Dependent Hypertension.

There are significant neurogenic and inflammatory influences on blood pressure, yet the role played by each of these processes in the development of hypertension is unclear. Tumor necrosis factor α (TNFα) has emerged as a critical modulator of blood pressure and neural plasticity; however, the mechanism by which TNFα signaling contributes to the development of hypertension is uncertain. We present evidence that following angiotensin II (AngII) infusion the TNFα type 1 receptor (TNFR1) plays a key role in heightened glutamate signaling in the hypothalamic paraventricular nucleus (PVN), a key central coordinator of blood pressure control. Fourteen day administration of a slow-pressor dose of AngII in male mice was associated with transcriptional and post-transcriptional (increased plasma membrane affiliation) regulation of TNFR1 in the PVN. Further, TNFR1 was shown to be critical for elevated NMDA-mediated excitatory currents in sympathoexcitatory PVN neurons following AngII infusion. Finally, silencing PVN TNFR1 prevented the increase in systolic blood pressure induced by AngII. These findings indicate that TNFR1 modulates a cellular pathway involving an increase in NMDA-mediated currents in the PVN following AngII infusion, suggesting a mechanism whereby TNFR1 activation contributes to hypertension via heightened hypothalamic glutamate-dependent signaling.SIGNIFICANCE STATEMENT Inflammation is critical for the emergence of hypertension, yet the mechanisms by which inflammatory mediators contribute to this dysfunction are not clearly defined. We show that tumor necrosis factor α receptor 1 (TNFR1) in the paraventricular hypothalamic nucleus (PVN), a critical neuroregulator of cardiovascular function, plays an important role in the development of hypertension in mice. In the PVN, TNFR1 expression and plasma membrane localization are upregulated during hypertension induced by angiotensin II (AngII). Further, TNFR1 activation was essential for NMDA signaling and the heightening NMDA currents during hypertension. Finally, TNFR1 silencing in the PVN inhibits elevated blood pressure induced by AngII. These results point to a critical role for hypothalamic TNFR1 signaling in hypertension.

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.

SorCS2 is required for social memory and trafficking of the NMDA receptor.

Social memory processing requires functional CA2 neurons, however the specific mechanisms that regulate their activity are poorly understood. Here, we document that SorCS2, a member of the family of the Vps10 family of sorting receptors, is highly expressed in pyramidal neurons of CA2, as well as ventral CA1, a circuit implicated in social memory. SorCS2 specifically localizes to the postsynaptic density and endosomes within dendritic spines of CA2 neurons. We have discovered that SorCS2 is a selective regulator of NMDA receptor surface trafficking in hippocampal neurons, without altering AMPA receptor trafficking. In addition, SorCS2 regulates dendritic spine density in CA2 neurons where SorCS2 expression is enriched, but not in dorsal CA1 neurons, which normally express very low levels of this protein. To specifically test the role of SorCS2 in behavior, we generated a novel SorCS2-deficient mouse, and identify a significant social memory deficit, with no change in sociability, olfaction, anxiety, or several hippocampal-dependent behaviors. Mutations in sorCS2 have been associated with bipolar disease, schizophrenia, and attention deficient-hyperactivity disorder, and abnormalities in social memory are core components of these neuropsychiatric conditions. Thus, our findings provide a new mechanism for social memory formation, through regulating synaptic receptor trafficking in pyramidal neurons by SorCS2.

Autocrine BDNF-TrkB signalling within a single dendritic spine.

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are crucial for many forms of neuronal plasticity, including structural long-term potentiation (sLTP), which is a correlate of an animal's learning. However, it is unknown whether BDNF release and TrkB activation occur during sLTP, and if so, when and where. Here, using a fluorescence resonance energy transfer-based sensor for TrkB and two-photon fluorescence lifetime imaging microscopy, we monitor TrkB activity in single dendritic spines of CA1 pyramidal neurons in cultured murine hippocampal slices. In response to sLTP induction, we find fast (onset < 1 min) and sustained (>20 min) activation of TrkB in the stimulated spine that depends on NMDAR (N-methyl-d-aspartate receptor) and CaMKII signalling and on postsynaptically synthesized BDNF. We confirm the presence of postsynaptic BDNF using electron microscopy to localize endogenous BDNF to dendrites and spines of hippocampal CA1 pyramidal neurons. Consistent with these findings, we also show rapid, glutamate-uncaging-evoked, time-locked BDNF release from single dendritic spines using BDNF fused to superecliptic pHluorin. We demonstrate that this postsynaptic BDNF-TrkB signalling pathway is necessary for both structural and functional LTP. Together, these findings reveal a spine-autonomous, autocrine signalling mechanism involving NMDAR-CaMKII-dependent BDNF release from stimulated dendritic spines and subsequent TrkB activation on these same spines that is crucial for structural and functional plasticity.

Acute Delta 9-tetrahydrocannabinol administration differentially alters the hippocampal opioid system in adult female and male rats.

Our prior studies demonstrated that the rat hippocampal opioid system can undergo sex-specific adaptations to external stimuli that can influence opioid-associated learning processes. This opioid system extensively overlaps with the cannabinoid system. Moreover, acute administration of Δ9 Tetrahydrocannabinoid (THC), the primary psychoactive constituent of cannabis, can alter cognitive behaviors that involve the hippocampus. Here, we use light and electron microscopic immunocytochemical methods to examine the effects of acute THC (5 mg/kg, i.p., 1 h) on mossy fiber Leu-Enkephalin (LEnk) levels and the distribution and phosphorylation levels of delta and mu opioid receptors (DORs and MORs, respectively) in CA3 pyramidal cells and parvalbumin dentate hilar interneurons of adult female and male Sprague-Dawley rats. In females with elevated estrogen states (proestrus/estrus stage), acute THC altered the opioid system so that it resembled that seen in vehicle-injected females with low estrogen states (diestrus) and males: (1) mossy fiber LEnk levels in CA2/3a decreased; (2) phosphorylated-DOR levels in CA2/3a pyramidal cells increased; and (3) phosphorylated-MOR levels increased in most CA3b laminae. In males, acute THC resulted in the internalization of MORs in parvalbumin-containing interneuron dendrites which would decrease disinhibition of granule cells. In both sexes, acute THC redistributed DORs to the near plasma membrane of CA3 pyramidal cell dendrites, however, the dendritic region varied with sex. Additionally, acute THC also resulted in a sex-specific redistribution of DORs within CA3 pyramidal cell dendrites which could differentially promote synaptic plasticity and/or opioid-associated learning processes in both females and males.

Oxycodone injections not paired with conditioned place preference have little effect on the hippocampal opioid system in female and male rats.

Oxycodone (Oxy) conditioned place preference (CPP) in Sprague Dawley rats results in sex-specific alterations in hippocampal opioid circuits in a manner that facilitates opioid-associative learning processes, particularly in females. Here, we examined if Oxy (3 mg/kg, I.P.) or saline (Sal) injections not paired with behavioral testing similarly affect the hippocampal opioid system. Sal-injected females compared to Sal-injected males had: (1) higher densities of cytoplasmic delta opioid receptors (DOR) in GABAergic hilar dendrites suggesting higher baseline reserve DOR pools and (2) elevated phosphorylated DOR levels, but lower phosphorylated mu opioid receptor (MOR) levels in CA3a suggesting that the baseline pools of activated opioid receptors vary in females and males. In contrast to CPP studies, Oxy-injections in the absence of behavioral tests resulted in few changes in the hippocampal opioid system in either females or males. Specifically, Oxy-injected males compared to Sal-injected males had fewer DORs near the plasma membrane of CA3 pyramidal cell dendrites and in CA3 dendritic spines contacted by mossy fibers, and lower pMOR levels in CA3a. Oxy-injected females compared to Sal-injected females had higher total DORs in GABAergic dendrites and lower total MORs in parvalbumin-containing dendrites. Thus, unlike Oxy CPP, Oxy-injections redistributed opioid receptors in hippocampal neurons in a manner that would either decrease (males) or not alter (females) excitability and plasticity processes. These results indicate that the majority of changes within hippocampal opioid circuits that would promote opioid-associative learning processes in both females and males do not occur with Oxy administration alone, and instead must be paired with CPP.

Correction: Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca2+ channels.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Cocaine- and stress-primed reinstatement of drug-associated memories elicit differential behavioral and frontostriatal circuit activity patterns via recruitment of L-type Ca2+ channels.

Cocaine-associated memories are critical drivers of relapse in cocaine-dependent individuals that can be evoked by exposure to cocaine or stress. Whether these environmental stimuli recruit similar molecular and circuit-level mechanisms to promote relapse remains largely unknown. Here, using cocaine- and stress-primed reinstatement of cocaine conditioned place preference to model drug-associated memories, we find that cocaine drives reinstatement by increasing the duration that mice spend in the previously cocaine-paired context whereas stress increases the number of entries into this context. Importantly, both forms of reinstatement require Cav1.2 L-type Ca2+ channels (LTCCs) in cells of the prelimbic cortex that project to the nucleus accumbens core (PrL→NAcC). Utilizing fiber photometry to measure circuit activity in vivo in conjunction with the LTCC blocker, isradipine, we find that LTCCs drive differential recruitment of the PrL→ NAcC pathway during cocaine- and stress-primed reinstatement. While cocaine selectively activates PrL→NAcC cells prior to entry into the cocaine-paired chamber, a measure that is predictive of duration in that chamber, stress increases persistent activity of this projection, which correlates with entries into the cocaine-paired chamber. Using projection-specific chemogenetic manipulations, we show that PrL→NAcC activity is required for both cocaine- and stress-primed reinstatement, and that activation of this projection in Cav1.2-deficient mice restores reinstatement. These data indicate that LTCCs are a common mediator of cocaine- and stress-primed reinstatement. However, they engage different patterns of behavior and PrL→NAcC projection activity depending on the environmental stimuli. These findings establish a framework to further study how different environmental experiences can drive relapse, and supports further exploration of isradipine, an FDA-approved LTCC blocker, as a potential therapeutic for the prevention of relapse in cocaine-dependent individuals.

Molecular profiling of reticular gigantocellularis neurons indicates that eNOS modulates environmentally dependent levels of arousal.

Neurons of the medullary reticular nucleus gigantocellularis (NGC) and their targets have recently been a focus of research on mechanisms supporting generalized CNS arousal (GA) required for proper cognitive functions. Using the retro-TRAP method, we characterized transcripts enriched in NGC neurons which have projections to the thalamus. The unique expression and activation of the endothelial nitric oxide (eNOS) signaling pathway in these cells and their intimate connections with blood vessels indicate that these neurons exert direct neurovascular coupling. Production of nitric oxide (NO) within eNOS-positive NGC neurons increases after environmental perturbations, indicating a role for eNOS/NO in modulating environmentally appropriate levels of GA. Inhibition of NO production causes dysregulated behavioral arousal after exposure to environmental perturbation. Further, our findings suggest interpretations for associations between psychiatric disorders and mutations in the eNOS locus.

Loss of APOBEC1 RNA-editing function in microglia exacerbates age-related CNS pathophysiology.

Microglia (MG), a heterogeneous population of phagocytic cells, play important roles in central nervous system (CNS) homeostasis and neural plasticity. Under steady-state conditions, MG maintain homeostasis by producing antiinflammatory cytokines and neurotrophic factors, support myelin production, and remove synapses and cellular debris, as well as participating in "cross-correction," a process that supplies neurons with key factors for executing autophagy-lysosomal function. As sentinels for the immune system, MG also detect "danger" signals (pathogenic or traumatic insult), become activated, produce proinflammatory cytokines, and recruit monocytes and dendritic cells to the site of damage through a breached blood-brain barrier or via brain lymphatics. Failure to effectively resolve MG activation can be problematic and can lead to chronic inflammation, a condition proposed to underlie CNS pathophysiology in heritable brain disorders and age-related neurodegenerative and cognitive decline. Here, we show that APOBEC1-mediated RNA editing occurs within MG and is key to maintaining their resting status. Like bone marrow-derived macrophages, RNA editing in MG leads to overall changes in the abundance of edited proteins that coordinate the function of multiple cellular pathways. Conversely, mice lacking the APOBEC1 editing function in MG display evidence of dysregulation, with progressive age-related signs of neurodegeneration, characterized by clustering of activated MG, aberrant myelination, increased inflammation, and lysosomal anomalies that culminate in behavioral and motor deficiencies. Collectively, our study identifies posttranscriptional modification by RNA editing as a critical regulatory mechanism of vital cellular functions that maintain overall brain health.

ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10S55L mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10S55L mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10S55L-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10S55L mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

Chronic immobilization stress primes the hippocampal opioid system for oxycodone-associated learning in female but not male rats.

In adult female, but not male, Sprague Dawley rats, chronic immobilization stress (CIS) increases mossy fiber (MF) Leu-Enkephalin levels and redistributes delta- and mu-opioid receptors (DORs and MORs) in hippocampal CA3 pyramidal cells and GABAergic interneurons to promote excitation and learning processes following subsequent opioid exposure. Here, we demonstrate that CIS females, but not males, acquire conditioned place preference (CPP) to oxycodone and that CIS "primes" the hippocampal opioid system in females for oxycodone-associated learning. In CA3b, oxycodone-injected (Oxy) CIS females relative to saline-injected (Sal) CIS females exhibited an increase in the cytoplasmic and total densities of DORs in pyramidal cell dendrites so that they were similar to Sal- and Oxy-CIS males. Consistent with our earlier studies, Sal- and Oxy-CIS females but not CIS males had elevated DOR densities in MF-CA3 dendritic spines, which we have previously shown are important for opioid-mediated long-term potentiation. In the dentate gyrus, Oxy-CIS females had more DOR-labeled interneurons than Sal-CIS females. Moreover, Sal- and Oxy-CIS females compared to both groups of CIS males had elevated levels of DORs and MORs in GABAergic interneuron dendrites, suggesting capacity for greater synthesis or storage of these receptors in circuits important for opioid-mediated disinhibition. However, more plasmalemmal MORs were on large parvalbumin-containing dendrites of Oxy-CIS males compared to Sal-CIS males, suggesting a limited ability for increased granule cell disinhibition. These results suggest that low levels of DORs in MF-CA3 synapses and hilar GABAergic interneurons may contribute to the attenuation of oxycodone CPP in males exposed to CIS.

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.

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage leads to disability or death with few established treatments. Adverse outcomes after intracerebral hemorrhage result from irreversible damage to neurons resulting from primary and secondary injury. Secondary injury has been attributed to hemoglobin and its oxidized product hemin from lysed red blood cells. The aim of this study was to identify the underlying cell death mechanisms attributable to secondary injury by hemoglobin and hemin to broaden treatment options. METHODS: We investigated cell death mechanisms in cultured neurons exposed to hemoglobin or hemin. Chemical inhibitors implicated in all known cell death pathways were used. Identified cell death mechanisms were confirmed using molecular markers and electron microscopy. RESULTS: Chemical inhibitors of ferroptosis and necroptosis protected against hemoglobin- and hemin-induced toxicity. By contrast, inhibitors of caspase-dependent apoptosis, protein or mRNA synthesis, autophagy, mitophagy, or parthanatos had no effect. Accordingly, molecular markers of ferroptosis and necroptosis were increased after intracerebral hemorrhage in vitro and in vivo. Electron microscopy showed that hemin induced a necrotic phenotype. Necroptosis and ferroptosis inhibitors each abrogated death by >80% and had similar therapeutic windows in vitro. CONCLUSIONS: Experimental intracerebral hemorrhage shares features of ferroptotic and necroptotic cell death, but not caspase-dependent apoptosis or autophagy. We propose that ferroptosis or necroptotic signaling induced by lysed blood is sufficient to reach a threshold of death that leads to neuronal necrosis and that inhibition of either of these pathways can bring cells below that threshold to survival.

Understanding the broad influence of sex hormones and sex differences in the brain.

Sex hormones act throughout the entire brain of both males and females via both genomic and nongenomic receptors. Sex hormones can act through many cellular and molecular processes that alter structure and function of neural systems and influence behavior as well as providing neuroprotection. Within neurons, sex hormone receptors are found in nuclei and are also located near membranes, where they are associated with presynaptic terminals, mitochondria, spine apparatus, and postsynaptic densities. Sex hormone receptors also are found in glial cells. Hormonal regulation of a variety of signaling pathways as well as direct and indirect effects on gene expression induce spine synapses, up- or downregulate and alter the distribution of neurotransmitter receptors, and regulate neuropeptide expression and cholinergic and GABAergic activity as well as calcium sequestration and oxidative stress. Many neural and behavioral functions are affected, including mood, cognitive function, blood pressure regulation, motor coordination, pain, and opioid sensitivity. Subtle sex differences exist for many of these functions that are developmentally programmed by hormones and by not yet precisely defined genetic factors, including the mitochondrial genome. These sex differences and responses to sex hormones in brain regions, which influence functions not previously regarded as subject to such differences, indicate that we are entering a new era of our ability to understand and appreciate the diversity of gender-related behaviors and brain functions. © 2016 Wiley Periodicals, Inc.

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.

Opioid receptor-dependent sex differences in synaptic plasticity in the hippocampal mossy fiber pathway of the adult rat.

The mossy fiber (MF) pathway is critical to hippocampal function and influenced by gonadal hormones. Physiological data are limited, so we asked whether basal transmission and long-term potentiation (LTP) differed in slices of adult male and female rats. The results showed small sex differences in basal transmission but striking sex differences in opioid receptor sensitivity and LTP. When slices were made from females on proestrous morning, when serum levels of 17ß-estradiol peak, the nonspecific opioid receptor antagonist naloxone (1 µm) enhanced MF transmission but there was no effect in males, suggesting preferential opioid receptor-dependent inhibition in females when 17ß-estradiol levels are elevated. The µ-opioid receptor (MOR) antagonist Cys2,Tyr3,Orn5,Pen7-amide (CTOP; 300 nm) had a similar effect but the δ-opioid receptor (DOR) antagonist naltrindole (NTI; 1 µm) did not, implicating MORs in female MF transmission. The GABAB receptor antagonist saclofen (200 µm) occluded effects of CTOP but the GABAA receptor antagonist bicuculline (10 µm) did not. For LTP, a low-frequency (LF) protocol was used because higher frequencies elicited hyperexcitability in females. Proestrous females exhibited LF-LTP but males did not, suggesting a lower threshold for synaptic plasticity when 17ß-estradiol is elevated. NTI blocked LF-LTP in proestrous females, but CTOP did not. Electron microscopy revealed more DOR-labeled spines of pyramidal cells in proestrous females than males. Therefore, we suggest that increased postsynaptic DORs mediate LF-LTP in proestrous females. The results show strong MOR regulation of MF transmission only in females and identify a novel DOR-dependent form of MF LTP specific to proestrus.

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.