Revisiting sex as a biological variable in hypertension research.

Half of adults in the United States have hypertension as defined by clinical practice guidelines. Interestingly, women are generally more likely to be aware of their hypertension and have their blood pressure controlled with treatment compared with men, yet hypertension-related mortality is greater in women. This may reflect the fact that the female sex remains underrepresented in clinical and basic science studies investigating the effectiveness of therapies and the mechanisms controlling blood pressure. This Review provides an overview of the impact of the way hypertension research has explored sex as a biological variable (SABV). Emphasis is placed on epidemiological studies, hypertension clinical trials, the genetics of hypertension, sex differences in immunology and gut microbiota in hypertension, and the effect of sex on the central control of blood pressure. The goal is to offer historical perspective on SABV in hypertension, highlight recent studies that include SABV, and identify key gaps in SABV inclusion and questions that remain in the field. Through continued awareness campaigns and engagement/education at the level of funding agencies, individual investigators, and in the editorial peer review system, investigation of SABV in the field of hypertension research will ultimately lead to improved clinical outcomes.

A Skeletal Muscle-Mediated Anticontractile Response on Vascular Tone: Unraveling the Lactate-AMPK-NOS1 Pathway in Femoral Arteries.

The regulation of vascular tone by perivascular tissues is a complex interplay of various paracrine factors. Here, we investigate the anti-contractile effect of skeletal muscle surrounding the femoral and carotid arteries and its underlying mechanisms. Using male and female Wistar rats, we demonstrated that serotonin, phenylephrine, and U-46619 induced a concentration-dependent vasoconstrictor response in femoral artery rings. Interestingly, this response was diminished in the presence of surrounding femoral skeletal muscle, irrespective of sex. No anti-contractile effect was observed when the carotid artery was exposed to its surrounding skeletal muscle. The observed effect in the femoral artery persisted even in the absence of endothelium and when the muscle was detached from the artery. Furthermore, the skeletal muscle surrounding the femoral artery was able to promote an anti-contractile effect in three other vascular beds (basilar, mesenteric, and carotid arteries). Using inhibitors of lactate dehydrogenase and the 1/4 monocarboxylate transporter, we confirmed the involvement of lactate, as both inhibitors were able to abolish the anti-contractile effect. However, lactate did not directly promote vasodilation; rather, it exerted its effect by activating 5' AMP-activated protein kinase (AMPK) and neuronal nitric oxide synthase (NOS1) in the skeletal muscle. Accordingly, Nω-propyl L-arginine, a specific inhibitor of NOS1, prevented the anti-contractile effect, as well as lactate-induced phosphorylation of NOS1 at the stimulatory serine site (1417) in primary skeletal muscle cells. Phosphorylation of NOS1 was reduced in the presence of Bay-3827, a selective AMPK inhibitor. In conclusion, femoral artery-associated skeletal muscle is a potent paracrine and endocrine organ that influences vascular tone in both sexes. Mechanistically, the anti-contractile effect involves muscle fiber type and/or its anatomical location but not the type of artery or its related vascular endothelium. Finally, the femoral artery anti-contractile effect is mediated by the lactate-AMPK-phospho-NOS1Ser1417-NO signaling axis.

Vascular dysfunction occurs prior to the onset of amyloid pathology and Aß plaque deposits colocalize with endothelial cells in the hippocampus of female APPswe/PSEN1dE9 mice.

Increasing evidence shows that cardiovascular diseases (CVDs) are associated with an increased risk of cognitive impairment and Alzheimer's diseases (AD). It is unknown whether systemic vascular dysfunction occurs prior to the development of AD, if this occurs in a sex-dependent manner, and whether endothelial cells play a role in the deposition of amyloid beta (Aß) peptides. We hypothesized that vascular dysfunction occurs prior to the onset of amyloid pathology, thus escalating its progression. Furthermore, endothelial cells from female mice will present with an exacerbated formation of Aß peptides due to an exacerbated pressure pulsatility. To test this hypothesis, we used a double transgenic mouse model of early-onset AD (APPswe/PSEN1dE9). We evaluated hippocampus-dependent recognition memory and the cardiovascular function by echocardiography and direct measurements of blood pressure through carotid artery catheterization. Vascular function was evaluated in resistance arteries, morphometric parameters in the aortas, and immunofluorescence in the hippocampus and aortas. We observed that endothelial dysfunction occurred prior to the onset of amyloid pathology irrespective of sex. However, during the onset of amyloid pathology, only female APP/PS1 mice had vascular stiffness in the aorta. There was elevated Aß deposition which colocalized with endothelial cells in the hippocampus from female APP/PS1 mice. Overall, these data showed that vascular abnormalities may be an early marker, and potential mediator of AD, but exacerbated aortic stiffness and pressure pulsatility after the onset of amyloid pathology may be associated with a greater burden of Aß formation in hippocampal endothelial cells from female but not male APP/PS1 mice.

Daily fluctuations in blood glucose with normal aging are inversely related to hippocampal synaptic mitochondrial proteins.

Defective brain glucose utilization is a hallmark of Alzheimer's disease (AD) while Type II diabetes and elevated blood glucose escalate the risk for AD in later life. Isolating contributions of normal aging from coincident metabolic or brain diseases could lead to refined approaches to manage specific health risks and optimize treatments targeted to susceptible older individuals. We evaluated metabolic, neuroendocrine, and neurobiological differences between young adult (6 months) and aged (24 months) male rats. Compared to young adults, blood glucose was significantly greater in aged rats at the start of the dark phase of the day but not during the light phase. When challenged with physical restraint, a potent stressor, aged rats effected no change in blood glucose whereas blood glucose increased in young adults. Tissues were evaluated for markers of oxidative phosphorylation (OXPHOS), neuronal glucose transport, and synapses. Outright differences in protein levels between age groups were not evident, but circadian blood glucose was inversely related to OXPHOS proteins in hippocampal synaptosomes, independent of age. The neuronal glucose transporter, GLUT3, was positively associated with circadian blood glucose in young adults whereas aged rats tended to show the opposite trend. Our data demonstrate aging increases daily fluctuations in blood glucose and, at the level of individual differences, negatively associates with proteins related to synaptic OXPHOS. Our findings imply that glucose dyshomeostasis may exacerbate metabolic aspects of synaptic dysfunction that contribute to risk for age-related brain disorders.

Brain mitochondria in behavior: more than a powerhouse.

Rosenberg and colleagues developed a miniaturized assay of mitochondrial content and energy transformation capacity that allowed them to assess mitochondrial features and activities across the brain in male mice. Their findings provide a comprehensive brain-wide map of mitochondrial associations with stress-induced behaviors and highlight mitochondria as dynamic, spatially-organized organelles.

A Novel Tissue-Specific Insight into Sex Steroid Fluctuations Throughout the Murine Estrous Cycle.

Serum sex steroid levels fluctuate throughout the reproductive cycle. However, the degree to which sex steroid tissue content mimics circulating content is unknown. Understanding the flux and physiological quantity of tissue steroid content is imperative for targeted hormonal therapy development. Utilizing a gold-standard ultrasensitive liquid chromatography-mass spectrometry (LC/MS) method we determined sex steroid (17ß-estradiol [E2], testosterone, androstenedione, and progesterone) fluctuations in serum and in 15 tissues throughout the murine estrous cycle (proestrus, estrus, and diestrus I) and in ovariectomized (OVX) mice. We observed dynamic fluctuations in serum and tissue steroid content throughout the estrous cycle with proestrus generally presenting the highest content of E2, testosterone, and androstenedione, and lowest content of progesterone. In general, the trend in circulating steroid content between the stages of the estrous cycle was mimicked in tissue. However, the absolute amounts of steroid levels when normalized to tissue weight were found to be significantly different between the tissues with the serum steroid quantity often being significantly lower than the tissue quantity. Additionally, we found that OVX mice generally displayed a depletion of all steroids in the various tissues assessed, except in the adrenal glands which were determined to be the main site of peripheral E2 production after ovary removal. This investigation provides a comprehensive analysis of steroid content throughout the estrous cycle in a multitude of tissues and serum. We believe this information will help serve as the basis for the development of physiologically relevant, tissue-specific hormonal therapies.

Gestational stress decreases postpartum mitochondrial respiration in the prefrontal cortex of female rats.

Postpartum depression (PPD) is a major psychiatric complication of childbirth, affecting up to 20% of mothers, yet remains understudied. Mitochondria, dynamic organelles crucial for cell homeostasis and energy production, share links with many of the proposed mechanisms underlying PPD pathology. Brain mitochondrial function is affected by stress, a major risk factor for development of PPD, and is linked to anxiety-like and social behaviors. Considering the importance of mitochondria in regulating brain function and behavior, we hypothesized that mitochondrial dysfunction is associated with behavioral alterations in a chronic stress-induced rat model of PPD. Using a validated and translationally relevant chronic mild unpredictable stress paradigm during late gestation, we induced PPD-relevant behaviors in adult postpartum Wistar rats. In the mid-postpartum, we measured mitochondrial function in the prefrontal cortex (PFC) and nucleus accumbens (NAc) using high-resolution respirometry. We then measured protein expression of mitochondrial complex proteins and 4-hydroxynonenal (a marker of oxidative stress), and Th1/Th2 cytokine levels in PFC and plasma. We report novel findings that gestational stress decreased mitochondrial function in the PFC, but not the NAc of postpartum dams. However, in groups controlling for the effects of either stress or parity alone, no differences in mitochondrial respiration measured in either brain regions were observed compared to nulliparous controls. This decrease in PFC mitochondrial function in stressed dams was accompanied by negative behavioral consequences in the postpartum, complex-I specific deficits in protein expression, and increased Tumor Necrosis Factor alpha cytokine levels in plasma and PFC. Overall, we report an association between PFC mitochondrial respiration, PPD-relevant behaviors, and inflammation following gestational stress, highlighting a potential role for mitochondrial function in postpartum health.

Mitochondrial might: powering the peripartum for risk and resilience.

The peripartum period, characterized by dynamic hormonal shifts and physiological adaptations, has been recognized as a potentially vulnerable period for the development of mood disorders such as postpartum depression (PPD). Stress is a well-established risk factor for developing PPD and is known to modulate mitochondrial function. While primarily known for their role in energy production, mitochondria also influence processes such as stress regulation, steroid hormone synthesis, glucocorticoid response, GABA metabolism, and immune modulation - all of which are crucial for healthy pregnancy and relevant to PPD pathology. While mitochondrial function has been implicated in other psychiatric illnesses, its role in peripartum stress and mental health remains largely unexplored, especially in relation to the brain. In this review, we first provide an overview of mitochondrial involvement in processes implicated in peripartum mood disorders, underscoring their potential role in mediating pathology. We then discuss clinical and preclinical studies of mitochondria in the context of peripartum stress and mental health, emphasizing the need for better understanding of this relationship. Finally, we propose mitochondria as biological mediators of resilience to peripartum mood disorders.

Aging or chronic stress impairs working memory and modulates GABA and glutamate gene expression in prelimbic cortex.

The glucocorticoid (GC) hypothesis posits that effects of stress and dysregulated hypothalamic-pituitary-adrenal axis activity accumulate over the lifespan and contribute to impairment of neural function and cognition in advanced aging. The validity of the GC hypothesis is bolstered by a wealth of studies that investigate aging of the hippocampus and decline of associated mnemonic functions. The prefrontal cortex (PFC) mediates working memory which also decreases with age. While the PFC is susceptible to stress and GCs, few studies have formally assessed the application of the GC hypothesis to PFC aging and working memory. Using parallel behavioral and molecular approaches, we compared the effects of normal aging versus chronic variable stress (CVS) on working memory and expression of genes that encode for effectors of glutamate and GABA signaling in male F344 rats. Using an operant delayed match-to-sample test of PFC-dependent working memory, we determined that normal aging and CVS each significantly impaired mnemonic accuracy and reduced the total number of completed trials. We then determined that normal aging increased expression of Slc6a11, which encodes for GAT-3 GABA transporter expressed by astrocytes, in the prelimbic (PrL) subregion of the PFC. CVS increased PrL expression of genes associated with glutamatergic synapses: Grin2b that encodes the GluN2B subunit of NMDA receptor, Grm4 that encodes for metabotropic glutamate receptor 4 (mGluR4), and Plcb1 that encodes for phospholipase C beta 1, an intracellular signaling enzyme that transduces signaling of Group I mGluRs. Beyond the identification of specific genes that were differentially expressed between the PrL in normal aging or CVS, examination of Log2 fold-changes for all expressed glutamate and GABA genes revealed a positive association between molecular phenotypes of aging and CVS in the PrL but no association in the infralimbic subregion. Consistent with predictions of the GC hypothesis, PFC-dependent working memory and PrL glutamate/GABA gene expression demonstrate comparable sensitivity to aging and chronic stress. However, changes in expression of specific genes affiliated with regulation of extracellular GABA in normal aging vs. genes encoding for effectors of glutamatergic signaling during CVS suggest the presence of unique manifestations of imbalanced inhibitory and excitatory signaling in the PFC.

Glutathione in the nucleus accumbens regulates motivation to exert reward-incentivized effort.

Emerging evidence is implicating mitochondrial function and metabolism in the nucleus accumbens in motivated performance. However, the brain is vulnerable to excessive oxidative insults resulting from neurometabolic processes, and whether antioxidant levels in the nucleus accumbens contribute to motivated performance is not known. Here, we identify a critical role for glutathione (GSH), the most important endogenous antioxidant in the brain, in motivation. Using proton magnetic resonance spectroscopy at ultra-high field in both male humans and rodent populations, we establish that higher accumbal GSH levels are highly predictive of better, and particularly, steady performance over time in effort-related tasks. Causality was established in in vivo experiments in rats that, first, showed that downregulating GSH levels through micro-injections of the GSH synthesis inhibitor buthionine sulfoximine in the nucleus accumbens impaired effort-based reward-incentivized performance. In addition, systemic treatment with the GSH precursor N-acetyl-cysteine increased accumbal GSH levels in rats and led to improved performance, potentially mediated by a cell-type-specific shift in glutamatergic inputs to accumbal medium spiny neurons. Our data indicate a close association between accumbal GSH levels and an individual's capacity to exert reward-incentivized effort over time. They also suggest that improvement of accumbal antioxidant function may be a feasible approach to boost motivation.

Neuroinflammation and Mitochondrial Dysfunction Link Social Stress to Depression.

Major depressive disorder is a debilitating mental illness and a leading cause of global disease burden. While many etiological factors have been identified, social stress is a highly prevalent causative factor for the onset of depression. Unfortunately, rates of depression continue to increase around the world, and the recent COVID-19 pandemic has further exacerbated this mental health crisis. Though several therapeutic strategies are available, nearly 50% of patients who receive treatment never reach remission. The exact mechanisms by which social stress exposure promotes the development of depression are unclear, making it challenging to develop novel and more effective therapeutics. However, accumulating evidence points to a role for stress-induced neuroinflammation, particularly in treatment-resistant patients. Moreover, recent evidence has expanded the concept of the pathogenesis of depression to mitochondrial dysfunction, suggesting that the combined effects of social stress on mitochondria and inflammation may synergize to facilitate stress-related depression. In this chapter, we review evidence for neuroinflammation and mitochondrial dysfunction in the pathogenesis of social stress-induced depression and discuss these in the context of novel therapeutic targets for the treatment of depression.

Astrocytic release of ATP through type 2 inositol 1,4,5-trisphosphate receptor calcium signaling and social dominance behavior in mice.

Brain mitochondrial function is critical for numerous neuronal processes. We recently identified a link between brain energy and social dominance, where higher levels of mitochondrial function resulted in increased social competitive ability. The underlying mechanism of this link, however, remains unclear. Here, we investigated the contribution of astrocytic release of adenosine triphosphate (ATP) through the type 2 inositol 1,4,5-triphosphate receptor to social dominance behavior. Mice lacking the type 2 inositol 1,4,5-triphosphate receptor were characterized for their social dominance behavior, as well as their performance on a nonsocial task, the Morris Water Maze. In parallel, we also examined mitochondrial function in the medial prefrontal cortex, nucleus accumbens, and hippocampus to investigate how deficiencies in astrocytic ATP could modulate overall mitochondrial function. While knockout mice showed similar competitive ability compared with their wild-type littermates, dominant knockout mice exhibited a significant delay in exerting their dominance during the initial encounter. Otherwise, there were no differences in anxiety and exploratory traits, spatial learning and memory, or brain mitochondrial function in either light or dark circadian phases. Our findings point to a marginal role of astrocytic ATP through IP3 R2 in social competition, suggesting that, under basal conditions, the neuronal compartment is predominant for social dominance exertion.

Therapeutic potential of glutathione-enhancers in stress-related psychopathologies.

The mammalian brain has high energy demands, which may become higher in response to environmental challenges such as psychogenic stress exposure. Therefore, efficient neutralization of reactive oxygen species that are produced as a by-product of ATP synthesis is crucial for preventing oxidative damage and ensuring normal energy supply and brain function. Glutathione (GSH) is arguably the most important endogenous antioxidant in the brain. In recent years, aberrant GSH levels have been implicated in different psychiatric disorders, including stress-related psychopathologies. In this review, we examine the available data supporting a role for GSH levels and antioxidant function in the brain in relation to anxiety and stress-related psychopathologies. Additionally, we identify several promising compounds that could raise GSH levels in the brain by either increasing the availability of its precursors or the expression of GSH-regulating enzymes through activation of Nuclear factor erythroid-2-related factor 2 (Nrf2). Given the high tolerability and safety profile of these compounds, they may represent attractive new opportunities to complement existing therapeutic manipulations against stress-related psychopathologies.

The glucocorticoid receptor in the nucleus accumbens plays a crucial role in social rank attainment in rodents.

Social hierarchy in social species is usually established through competitive encounters with conspecifics. It determines the access to limited resources and, thus, leads to reduced fights among individuals within a group. Despite the known importance of social rank for health and well-being, the knowledge about the processes underlying rank attainment remains limited. Previous studies have highlighted the nucleus accumbens (NAc) as a key brain region in the attainment of social hierarchies in rodents. In addition, glucocorticoids and the glucocorticoid receptor (GR) have been implicated in the establishment of social hierarchies and social aversion. However, whether GR in the NAc is involved in social dominance is not yet known. To address this question, we first established that expression levels of GR in the NAc of high anxious, submissive-prone rats are lower than that of their low anxious, dominant-prone counterparts. Furthermore, virally-induced downregulation of GR expression in the NAc in rats led to an improvement of social dominance rank. We found a similar result in a cell-specific mouse model lacking GR in dopaminoceptive neurons (i.e., neurons containing dopamine receptors). Indeed, when cohabitating in dyads of mixed genotypes, mice deficient for GR in dopaminoceptive neurons had a higher probability to become dominant than wild-type mice. Overall, our results highlight GR in the NAc and in dopaminoceptive neurons as an important regulator of social rank attainment.

Disruption of mTOR and MAPK pathways correlates with severity in idiopathic autism.

The molecular signature underlying autism spectrum disorder remains largely unknown. This study identifies differential expression of mTOR and MAPK pathways in patients affected by mild and severe idiopathic autism. A total of 55 subjects were enrolled, of which 22 were typically developing individuals and 33 were patients aged between 3 and 11 years, with autism spectrum disorder. A detailed history, including physical examination, developmental evaluation, mental health history and autism diagnostic observation schedule were performed for each patient. Components of the mTOR and MAPK signalling pathways were analysed from peripheral blood at the protein level. Patients were then stratified according to their clinical phenotypes, and the molecular profiling was analysed in relation to the degree of autism severity. In this cohort of patients, we identified increased activity of mTOR and the MAPK pathways, key regulators of synaptogenesis and protein synthesis. Specifically, rpS6, p-eIF4E, TSC1 and p-MNK1 expression discriminated patients according to their clinical diagnosis, suggesting that components of protein synthesis signalling pathways might constitute a molecular signature of clinical severity in autism spectrum disorder.

Medium chain triglyceride diet reduces anxiety-like behaviors and enhances social competitiveness in rats.

Medium-chain triglycerides (MCT) are emerging as unique dietary supplements that are potentially relevant for the amelioration of brain dysfunctions. MCT are converted into ketones and free medium chain fatty acids that, in the brain, are highly effective energy sources to mitochondria and potentially less harmful than glucose metabolism to neurons. Given the recently established link between mitochondrial dysfunction and high anxiety and depression, we performed this study to investigate the effectiveness of an MCT-enriched diet to ameliorate anxiety- and depression-related behaviors in rats. Male rats were distributed into groups, according to their anxiety-like behaviors in the elevated plus maze. Each group was given either MCT-supplemented diet or an isocaloric control diet for fifteen days. Starting from the eighth day of diet, rats were exposed to different behavioral tests. MCT-fed rats exhibited reduced anxiety-like behaviors and enhanced social competitiveness, while their coping responses in the forced swim test were not affected by the treatment. When evaluated at the end of the two-week MCT diet, mitochondrial respiration was reduced in the medial prefrontal cortex (mPFC) while unchanged in the nucleus accumbens. In the mPFC, enzymes related to glycolysis and oxidative phosphorylation were also decreased by MCT diet, while proteins controlling glucose and glutamate transport were increased. Altogether, our findings strongly suggest the effectiveness of MCT diet to exert anxiolytic effects. In the brain, our results point to the mPFC as a brain region in which MCT supplementation improves transport and control of energy substrates.

Mitochondrial dysfunction in Autism Spectrum Disorder: clinical features and perspectives.

Autism Spectrum Disorder (ASD) is a prototypic pervasive developmental disorder characterized by social interaction, and communication deficits, repetitive, stereotypic patterns of behavior, and impairments in language and development. Clinical studies have identified mitochondrial disturbances at the levels of DNA, activity, complexes, oxidative stress, and metabolites in blood and urine of ASD patients. However, these observations from postmortem brains or peripheral tissues do not provide a direct link between autism and mitochondria. The synaptic abnormality of autistic patients has not been investigated yet. Here we review the findings of clinical studies investigating mitochondrial involvement in ASD patients, focusing particularly on the brain and the limitations and future directions needed in order to fully understand the role of mitochondria in ASD pathology.

Involvement of CRFR1 in the Basolateral Amygdala in the Immediate Fear Extinction Deficit.

Several animal and clinical studies have highlighted the ineffectiveness of fear extinction sessions delivered shortly after trauma exposure. This phenomenon, termed the immediate extinction deficit, refers to situations in which extinction programs applied shortly after fear conditioning may result in the reduction of fear behaviors (in rodents, frequently measured as freezing responses to the conditioned cue) during extinction training, but failure to consolidate this reduction in the long term. The molecular mechanisms driving this immediate extinction resistance remain unclear. Here we present evidence for the involvement of the corticotropin releasing factor (CRF) system in the basolateral amygdala (BLA) in male Wistar rats. Intra-BLA microinfusion of the CRFR1 antagonist NBI30775 enhances extinction recall, whereas administration of the CRF agonist CRF6-33 before delayed extinction disrupts recall of extinction. We link the immediate fear extinction deficit with dephosphorylation of GluA1 glutamate receptors at Ser845 and enhanced activity of the protein phosphatase calcineurin in the BLA. Their reversal after treatment with the CRFR1 antagonist indicates their dependence on CRFR1 actions. These findings can have important implications for the improvement of therapeutic approaches to trauma, as well as furthering our understanding of the neurobiological mechanisms underlying fear-related disorders.

Mitochondrial function in the brain links anxiety with social subordination.

Dominance hierarchies are integral aspects of social groups, yet whether personality traits may predispose individuals to a particular rank remains unclear. Here we show that trait anxiety directly influences social dominance in male outbred rats and identify an important mediating role for mitochondrial function in the nucleus accumbens. High-anxious animals that are prone to become subordinate during a social encounter with a low-anxious rat exhibit reduced mitochondrial complex I and II proteins and respiratory capacity as well as decreased ATP and increased ROS production in the nucleus accumbens. A causal link for these findings is indicated by pharmacological approaches. In a dyadic contest between anxiety-matched animals, microinfusion of specific mitochondrial complex I or II inhibitors into the nucleus accumbens reduced social rank, mimicking the low probability to become dominant observed in high-anxious animals. Conversely, intraaccumbal infusion of nicotinamide, an amide form of vitamin B3 known to enhance brain energy metabolism, prevented the development of a subordinate status in high-anxious individuals. We conclude that mitochondrial function in the nucleus accumbens is crucial for social hierarchy establishment and is critically involved in the low social competitiveness associated with high anxiety. Our findings highlight a key role for brain energy metabolism in social behavior and point to mitochondrial function in the nucleus accumbens as a potential marker and avenue of treatment for anxiety-related social disorders.