Sex-steroid-dependent plasticity of brain-stem autonomic circuits

Abstract

In the central nervous system (CNS), nuclei of the brain stem play a critical role in the integration of peripheral sensory information and the regulation of autonomic output in mammalian physiology. The nucleus tractus solitarius of the brain stem acts as a relay center that receives peripheral sensory input from vagal afferents of the nodose ganglia, integrates information from within the brain stem and higher central centers, and then transmits autonomic efferent output through downstream premotor nuclei, such as the nucleus ambiguus, the dorsal motor nucleus of the vagus, and the rostral ventral lateral medulla. Although there is mounting evidence that sex and sex hormones modulate autonomic physiology at the level of the CNS, the mechanisms and neurocircuitry involved in producing these functional consequences are poorly understood. Of particular interest in this review is the role of estrogen, progesterone, and 5α-reductase-dependent neurosteroid metabolites of progesterone (e.g., allopregnanolone) in the modulation of neurotransmission within brain-stem autonomic neurocircuits. This review will discuss our understanding of the actions and mechanisms of estrogen, progesterone, and neurosteroids at the cellular level of brain-stem nuclei. Understanding the complex interaction between sex hormones and neural signaling plasticity of the autonomic nervous system is essential to elucidating the role of sex in overall physiology and disease.

INTRODUCTION

With the introduction of the National Institutes of Health’s policies on rigor and reproducibility, a new emphasis has been placed on understanding sex and thereby sex hormones as biological variables. For years, it was assumed that fluctuations in sex hormones associated with the ovarian cycle produced significant and ubiquitous variability in physiological parameters. Therefore, studying males exclusively allowed for reductions in physiological variability. This classic assumption was recently challenged by the identification that some experimental outcomes exhibit greater interindividual differences in males than interindividual differences in females (12). There is even an argument to be made that males represent a population more susceptible to disease involving dysregulated autonomic signaling, such as cardiovascular disease (79) and metabolic syndrome (73). In these latter cases, the opposite sex is likely an excellent model since females exhibit a protective mechanism against autonomic dysfunction that could be harnessed as a therapeutic. Therefore, more investigations are needed to understand how sex and sex hormones are affecting brain-stem autonomic signaling. This is particularly striking in the context of synaptic plasticity and the use of whole cell patch-clamping, which recently received renewed attention for the technique’s ability to investigate the intricacies of neuronal function (93). Perhaps not surprising then, recent reports using these experimental approaches provide clear evidence that sex hormones do have actions within fundamental signaling units of the autonomic nervous system (56, 62, 65).

FRAMEWORK FOR THE INVESTIGATION OF SEX AND REPRODUCTIVE HORMONES IN AUTONOMIC NEUROPHYSIOLOGY

A brief discussion of experimental approaches used to determine sex differences is first presented here to provide a framework for sex differences in autonomic control (Fig. 1). However, those interested in a detailed discussion of experimental approaches should be directed to other reviews (11, 74). Sex differences are generally classified into two categories: organizational and activational. Activational differences are mediated by direct activity of sex steroids and are considered transient in nature, meaning that the presence of a steroid causes a reversible change. Investigations into sex differences may be started by simply comparing males and females. Often, however, sex differences are subtle, and, therefore, it is useful to separate females by their stage within the ovarian cycle. If these approaches indicate significant differences between males and females, follow-up experiments might include gonadectomized animals. If differences are abolished by gonadectomy, then gonadectomy with specific hormone supplementation can be used to determine which steroid is responsible for suspected activational differences.

Fig. 1.

A flowchart summarizing the framework of different experimental approaches and models used to determine sex differences. To determine whether a sex difference is present and to differentiate between an activational or organizational sex difference, a series of questions must be asked. After determining an effect of interest, a logical first question is to ask whether the effect varies between males and females. If so, one should proceed to comparing gonadectomized males and females. If the difference in effect disappears after males and females are gonadectomized, then this suggests an activational sex difference exists for which the cause may then be probed by hormone supplementation. If the difference in effect remains regardless of gonadectomy (GDX), then this suggests an organizational difference that must be tested using a method such as four-core genotype. Alternatively, one might be interested in whether the estrous cycle affects the responses of females to a known measurement. In this case, females would first be examined across different stages of the menstrual/estrous cycle. It would then be necessary to conduct an experiment in which gonadectomized females received hormone supplementation to mimic the different estrous stages and determine which stage correlates with the effect. Finally, if there is no difference in effect between males and females whether intact or gonadectomized, then the conclusion can be made that there is no sex difference.

If gonadectomized males and females maintain their differences in a measured outcome, then the role of organizational differences must be considered. Organizational differences are permanent in nature. These differences are mediated by chromosomal differences between the sexes (i.e., XX vs. XY). The best example of this might be the testicular testosterone surge that occurs early in perinatal development (117). The presence of a Y chromosome confers the sex-determining region Y (Sry) gene that is critical for the development of testes. Embryonic testes release testosterone during critical periods in development. This surge in testosterone has been suggested to masculinize the brain by causing permanent changes in neural circuitry (3, 67). Although the activity of a steroid is required, suggestive of an activational event, this surge leads to permanent changes in neuronal circuits even when testosterone levels return to normal. Therefore, the masculinization of the brain is classified as an organizational change. This masculinization of the brain is suggested as a mechanism for sex differences in postnatal development and maturation of the autonomic nervous system, especially its regulation of heart rate (15, 18) likely through differences in synaptic signaling (34). Organizational differences are best investigated using genetic model systems. One in particular, known as the four-core genotype, removes the Sry gene from the Y chromosome and places it on an autosomal chromosome (2). This model creates mice with the traditional sex chromosome to gonad development (i.e., XX with ovaries and XY with testes). However, the movement of the Sry gene to an autosomal chromosome creates two additional mice, XX with testes and XY with ovaries. Additional effects of sex chromosome “dosing” can be examined with models like the sex chromosome trisomy model (84).

Regardless of model(s) used, the interpretation of sex steroid signaling in any physiological system is challenging. The nuances associated with cycling hormone levels are difficult to model, especially since these hormones often interact with each other in a brain-region-specific way (66). This phenomenon has been confirmed with estrogen and progesterone in autonomic centers for hormone receptor expression (53, 77). Therefore, experimental approaches such as ovariectomy with single-hormone replacement can be seen as too reductionist, and genetic manipulations, to test activational and/or organizational effects, may have compensatory responses that complicate the interpretations and may not appropriately mimic normal physiological hormone activity. Importantly, it is possible for sex-specific adaptions to prevent overt changes, thus effectively “canceling each other out” (33). This potentially leads to the conclusion that no sex difference exists in a particular experimental outcome. However, these difficulties should not limit enthusiasm for this type of work since increasing our understanding of sex differences could lead to novel therapeutics for diseases associated with these systems.

SEX DIFFERENCES IN AUTONOMIC PHYSIOLOGY

The relationship between sex and autonomic regulation is complex (8, 57, 91) and highly dependent on age (8). For example, young women show a significantly blunted blood pressure response to autonomic blockade using the nicotinic receptor antagonist trimethaphan compared with young male counterparts (26), likely, in part, because young women lack the same relationship between mean sympathetic nerve activity and total peripheral resistance as men (50). However, as women age, these relationships skew toward more masculinized responses (8). These age-related changes are thought to mediate some of the sex differences in cardiovascular disease (i.e., young women show protection against cardiovascular diseases, yet there is a striking uptick in disease rates with menopause; Ref. 86). This decreased sympathetic tone is not limited to cardiovascular autonomic regulation; the counterregulatory response (CRR) to hypoglycemia is also blunted in women in part through blunted sympathetic activation (32, 98). However, complete autonomic blockade with trimethaphan significantly altered the CRR in women (51), indicating that the parasympathetic system likely plays a larger role compared with men. This conclusion is supported by preclinical models (59). Despite these sex differences in the CRR, there is no sex difference in the occurrence of hypoglycemia in patients with type 1 diabetes (32a), suggesting that women are likely protected from the effects of antecedent hypoglycemia on autonomic activity (32). Gastrointestinal function and disease is also markedly different between sexes and across the menstrual cycle (57) with notable differences in metabolic responses, including the use of different energy sources under normal, healthy conditions and during disease (72). Despite our understanding of the existence of sex differences throughout different autonomic functions, the neuromechanistic underpinning(s) of these differences is largely unexplored. Therefore, understanding the action of sex hormones on the regulation of autonomic circuits will be informative in our development of therapeutics to treat a wide array of autonomic dysfunction(s).

BRAIN-STEM AUTONOMIC CIRCUITS

Viscerosensory input from various peripheral organs synapse within the brain stem’s nucleus tractus solitarius (NTS; Fig. 2). These sensory afferent fibers use glutamate as their primary neurotransmitter to carry a wide array of physiological information, including baroreceptors and chemoreceptors from the carotid bodies/aortic arch and mechanoreceptors and chemoreceptors from the gut. Serving as a relay station, the first-order NTS neurons integrate and encode the sensory information received from viscerosensory afferent terminals and send this information to upstream central nuclei, like the hypothalamus’s paraventricular nucleus, or to downstream autonomic motor nuclei, like the dorsal motor nucleus of the vagus (DMV), nucleus ambiguus (NA), and ventral lateral medulla (VLM). Both the DMV and NA comprise the motor limb of the parasympathetic nervous system. The NA is the ventral parasympathetic premotor nucleus that sends prominent projections to the heart (75) and is critical for baroreflex responses (25) and respiratory sinus arrhythmia (80). The DMV is the dorsal parasympathetic premotor nucleus that sends prominent projections to subdiaphragmatic viscera, including the stomach, intestine, liver, and arguably the pancreas. Both of these nuclei are cholinergic and receive glutamatergic and GABAergic projections (7, 19, 31, 41, 64, 87, 116), including direct inputs from the NTS (19, 41, 42, 64). Therefore, these motor neurons are significantly regulated by synaptic input, including vagal afferent terminals via second-order NTS neurons or possibly through direct afferent connections (94). In the generation of the baroreflex (a critical cardiovascular regulatory mechanism), NTS neurons project to the caudal (C)VLM, which, in turn, projects to the rostral (R)VLM. The RVLM sends prominent projections to the intermediolateral cell column of the spinal cord, and, therefore, these cells serve as premotor sympathetic neurons.

Fig. 2.

A schematic representation of the autonomic brain stem. Viscerosensory afferent neurons from vital organs, such as the heart, gastrointestinal system, pancreas, adrenal glands, kidneys, and blood vessels, synapse in the nucleus tractus solitarius (NTS). NTS neurons modulate descending autonomic output nuclei, like the dorsal motor nucleus of the vagus (DMV), nucleus ambiguus (NA), and the ventral lateral medulla (VLM). Although significant work must be done to determine the integrated nature of sex steroid modulation of autonomic signaling, all critical autonomic brain-stem regions express estrogen and progesterone receptors. Of recent interest is allopregnanolone, a metabolite of progesterone, which may be produced locally by the NTS. The extent of allopregnanolone’s role in modulating signals is unknown, but there is evidence that it is important in the regulation of GABA_A receptor activity. Since GABAergic signaling is a key mechanism by which the NTS regulates DMV, rostral VLM (RVLM), and NA activity (and, in turn, peripheral organ system function), allopregnanolone could be a novel modulator of autonomic output. ALLO, allopregnanolone; AP, area postrema; CVLM, caudal VLM; DVC, dorsal vagal complex; ER, estrogen receptor; GPER, G protein-coupled estrogen receptor; IML, intermediolateral column; PR, progesterone receptor.

However, the autonomic brain stem does not exclusively rely on intact afferent connections for sensing the internal milieu. The NTS is part of a larger structure known as the dorsal vagal complex. In addition to the NTS, the dorsal vagal complex comprises area postrema and the DMV. Although the lipophilic nature of cholesterol-based steroid hormones allows them to readily penetrate the blood-brain barrier, area postrema is a circumventricular organ and resides outside of the blood-brain barrier. The fenestrated capillaries within the NTS allow for many types of large molecules to pass unimpeded (47), and indeed neurons within both area postrema and the NTS are capable of intrinsically sensing peripheral homeostatic signals (9, 13, 16, 82, 95, 118). Therefore, the dorsal vagal complex is a prime candidate to integrate sex hormone information into autonomic function with limited need for active/supported transport or high levels of steroid. Despite our current understanding of these circuits, all of this signaling circuitry undergoes robust experience-dependent plasticity (17, 24, 35) and more importantly may be uniquely susceptible to autonomic insults (35). Our limited understanding of the dynamic modulation of these autonomic brain-stem regions limits our potential to develop treatments to target these networks. Therefore, although brain-stem circuitry is critical in the regulation of multiple physiological homeostatic functions, significantly more work must be done to understand how sex and sex differences affect brain-stem autonomic signaling plasticity.

INFLUENCE OF REPRODUCTIVE HORMONES ON BRAIN-STEM AUTONOMIC CIRCUITS

Central Estrogen Receptors in Autonomic Physiology

Despite significant evidence for the role of sex and sex hormones in autonomic physiology, the mechanism(s) and neurocircuitry responsible for these differences are not well characterized under either healthy or disease conditions. Estrogen is a well-established mediator of activational sex differences. Traditionally, estrogens influence “long-term” signaling through two different nuclear estrogen receptors (ERα and ERβ) that serve as ligand-dependent transcriptional factors regulating gene expression (81). Emerging evidence also exists for a short-term or “rapid effect” of estrogen signaling. This type of signaling is thought to come, in part, from ERα and ERβ located at the plasma membrane (49) but also from a G protein-coupled estrogen receptor known as GPER (89). Both ERα and ERβ are expressed throughout the brain stem’s autonomic regulatory centers with prominent expression in the NTS (76, 108, 121) and RVLM (109) and exhibit sex differences in their expression patterns (101, 114). Although only a limited amount of research exists on GPER activity in brain-stem autonomic neurons, GPER expression has been identified in the NTS and NA (21, 78).

A well-established modulatory role for estrogen in the brain stem is illustrated by its impact on satiety signaling. Estrogen decreases food intake and thereby weight through an increased facilitation of satiety signaling (36), including an increased satiating potency of lipids (4); this effect correlates with increased c-fos expression in the NTS with lipid consumption, thereby suggesting the NTS as estrogen’s site of action (4, 37). Estradiol microinjections directly into the NTS increased the effectiveness of CCK to suppress feeding (111), whereas knockdown of ERα blunts CCK-dependent signaling (44). Estrogen also increases the density (27) and excitability (90) of vagal afferent terminals within the NTS, which are required for CCK-dependent satiation. Together, these data support the hypothesis that estrogen potentiates CCK-dependent vagal afferent signaling in the NTS and can modulate further information processing to upstream nuclei through its effect on the dorsal vagal complex. Estrogen’s anorectic actions are not limited to modulation of CCK signaling but also include other satiation factors like apolipoprotein A-IV (106) and leptin (28). For example, estrogen’s effects on apolipoprotein A-IV-dependent satiation require recruitment of steroid receptor coactivator-1 in the dorsal vagal complex (104) by ERα (105).

Estrogen’s modulation of vagally mediated homeostasis is also not limited to satiation (52). Central estrogen administration increases both vagal nerve activity and baroreflex sensitivity, and antagonizing ERα within the NA abolished this estrogen-induced increase in baroreflex sensitivity (96, 97). Similarly, microinjections of GPER agonists into the NA induce a bradycardia (22), suggesting that estrogen might work through multiple different receptor types to influence parasympathetic control of heart rate. Central estrogen administration, however, also decreases sympathetic tone (96, 97). With the use of comparisons between male and female rats, females have shown reduced sympathetic nerve activation after experimentally induced hypertension (120), and this protective effect in females can be abolished by knockdown of ERβ within the RVLM (107, 122). Taken together, these data indicate that estrogen receptors modulate autonomic responses via brain-stem circuitry through a complex interaction between multiple receptor types and different cellular locations, rather than just direct effects on afferent terminals and/or their synaptic connections within the NTS.

Despite evidence implicating estrogen in the modulation of autonomic responses, only a limited number of studies have been done to determine the direct mechanistic actions of estrogen on autonomic neuronal excitability. In nonautonomic brain regions, estrogen typically activates L-type voltage-gated calcium channels, resulting in an increase in neuronal excitability (100). With the use of comparisons between males and females in different ovarian cycle stages, estrogen has been suggested to inhibit DMV motor neuron firing (56), and direct estrogen application also inhibited the firing frequency of unidentified NTS neurons (121). A similar decrease in excitability exists after estrogen application in the RVLM through a reduction in L-type calcium channel number (115), whereas sex differences in sympathetic nerve activity after the induction of experimental hypertension (120) have been suggested to be mediated through sex differences in the postsynaptic membrane expression of excitatory glutamatergic receptors (113). Whether the mechanism(s) of action is on L-type calcium channels or glutamate receptors (or both), estrogen likely decreases RVLM neuron activity. Conversely, GPER activation depolarizes cardiac vagal motor neurons within the NA (22), and differential effects of estrogen receptors on calcium responses have been shown in area postrema (83). These data together suggest that the brain stem might be unique in estrogen mechanism(s) of action and/or in its number of nuclei in which estrogen reduces neuronal excitability. Moreover, the autonomic brain stem contains considerable cell-type-specific estrogen actions.

Central Progesterone Receptors in Autonomic Physiology

Although progesterone receptors were first discovered in the brain in 1973 (99), the direct action of progesterone receptors in neuronal signaling plasticity is significantly understudied compared with estrogen. Traditionally, transcriptional actions of progesterone occur through progesterone receptors with two isoforms (PR-A and PR-B) that are derived from distinct promotor regions of a single gene (29, 60). However, similar to estrogen, progesterone also has rapid, gene expression-independent signaling actions. Several diverse receptor types have been suggested to mediate the rapid, nontranscriptional actions of progesterone. Some of these include unique membrane-bound progesterone receptors (mPR; Ref. 123), classic PR-A/B receptors that also activate intracellular signaling pathways, and even progesterone binding directly to oxytocin receptors (46). PR-A/B receptors are expressed in several brain-stem regions, including the NTS and DMV (40, 61). Although, to our knowledge, no investigations have looked for their presence in the brain stem, mPRs have been identified in at least the hypothalamus (6). Given the diverse range of putative mediators of progesterone’s nontranscriptional actions, the suggested intercellular signaling cascades are equally diverse. However, significant evidence exists that mPRs ultimately converge on intracellular calcium stores for short-term effects (6). Although these effects remain to be studied in the brain stem, the ability of progesterone receptors to mobilize calcium in other tissues does suggest an ability to modulate neuronal activity (103).

A role for progesterone in the brain stem has not been shown for modulation of cardiovascular function, but progesterone appears to regulate brain-stem control of respiratory function. The importance of the progesterone in respiratory function was first identified when progesterone microinjection into the NTS produced significant, dose-dependent increases in respiratory frequency that were blocked when progesterone receptors were antagonized (10). However, the neuronal substrate for this progesterone-receptor-dependent hyperventilation remains unclear and likely is state-dependent. For example, progesterone did not affect NTS neuronal discharge under normal conditions (112) but did blunt NTS neuronal responses to hypoxia (85). In addition, since the magnitude of hyperventilation is increased in the presence of estrogen, suggesting that estrogen can increase the gain of progesterone-induced hyperventilation (23, 55). Since respiratory centers in the brain stem contribute to cardiorespiratory coupling (20, 80), it is likely that progesterone modulates cardiovascular autonomic regulation in some form through its effects on brain-stem circuitry. Additionally, given the importance of oxytocin receptor signaling in the dorsal vagal complex (87, 88), the ability of progesterone receptors to translocate oxytocin receptors to the membrane in the hypothalamus remains a potential mechanism of action of progesterone receptors in the brain stem (102).

Central Allopregnanolone in Autonomic Physiology

Progesterone has also been implicated in an additional type of neuromodulation through its catalyzation by 5α-reductase into the neurosteroid, allopregnanolone (39, 69). Traditionally, neurosteroids are considered positive allosteric modulators of the γ-aminobutyric-A (GABA_A) receptors responsible for fast inhibitory neurotransmission (39), and their actions on GABA_A receptor signaling have been established in several regions of the brain, including the hypothalamus (38). Many autonomic brain-stem nuclei, including the NA (14), NTS (42, 45), DMV (7, 31, 43), and RVLM (30, 63), are tightly regulated by GABA_A receptor-mediated inhibition, and allopregnanolone is known to concentrate in the brain stem in both males and females (110). Allopregnanolone increases inhibition of hippocampal neurons across the estrous cycle and during pregnancy (68, 70, 119), and similar changes in GABA_A receptor inhibition have been demonstrated in the brain stem, at least for the DMV (65). The effects of allopregnanolone on respiratory circuits have been confirmed through GABA_A receptors (92). Therefore, these endogenous modulators of GABAergic inhibition may play a critical role in regulating both autonomic nervous system function and dysfunction.

In addition to identifying a progesterone-receptor-dependent facilitation of respiratory drive, the seminal work by Bayliss et al. (10) also identified a significant drop in blood pressure that was not eliminated by progesterone receptor antagonism. Follow-up investigations have since suggested that these effects are mediated not by progesterone receptor activation but through progesterone’s catalyzation to allopregnanolone (71). This allopregnanolone-induced decrease in blood pressure associates with a blunted baroreflex response (54, 71) through a brain-stem pathway (54). Since exogenous allopregnanolone application attenuates vagal afferent transmission in NTS neurons through GABA_A receptor activation, which results in decreased afferent-stimulation-induced firing within NTS neurons (62), allopregnanolone-mediated inhibition of afferent signaling is a potential mechanism for allopregnanolone’s blunting of the baroreflex. In addition to direct effects on GABA_A receptors, 5α-reductase-dependent neurosteroids increase membrane insertion of GABA_A receptors (1). Although this has not been confirmed as a functional change resulting from neurosteroid activity, a similar increase in membrane insertion of GABA_A receptors has been proposed for diabetes-induced plasticity of GABAergic neurotransmission in DMV neurons (17). Allopregnanolone may also exert opposing effects on postsynaptic GABA_A receptor activity depending on extracellular GABA concentration (48), suggesting that further work is needed to determine the complex interplay between allopregnanolone and GABA_A receptors in autonomic circuits.

Although allopregnanolone readily and rapidly crosses the blood-brain barrier when injected into the systematic circulation (58), evidence also exists for its local production within the brain (39). The notion of locally produced allopregnanolone is supported by the observation that a progesterone-mediated decrease in blood pressure was not blocked by the antagonism of progesterone since this experiment would require the conversion of progesterone to allopregnanolone locally (10). Together, these data support the NTS as a likely location of progesterone conversion to allopregnanolone. Although we do not currently know the exact location of 5α-reductase activity within the brain stem, there is a compelling case for the role of allopregnanolone in the regulation of at least parasympathetic nervous system output and reflex responses.

CONCLUSIONS

The new National Institute of Health's emphasis on understanding sex as a biological variable has promoted the need to understand its role in autonomic control. Despite abundant evidence that sex plays a role in signal processing within autonomic brain-stem circuits, considerable research must be done to further our understanding of mechanism(s) of action for regulating many aspects of physiology, including neural excitability of autonomic brain circuitry. Given the prominent sex differences in physiological responses and diseases that are regulated by the autonomic nervous system, these types of investigations are critical to our development of therapeutics for diseases, including those associated with the cardiovascular and metabolic system. In terms of effects of major sex steroids, estrogen, progesterone, and the progesterone derivative allopregnanolone are implicated in the regulation of neural communication within the autonomic brain stem. Specifically, current evidence suggests that estrogen promotes vagally mediated responses (i.e., satiation and baroreflex) while restraining sympathetic drive. It does this through a wide array of effects on neuronal excitability within individual autonomic brain-stem regions, which likely depend on whether a region promotes or inhibits vagal responses. Progesterone, on the other hand, has received limited attention, making it difficult to assess its role in overall autonomic drive. However, progesterone receptors are present within key autonomic nuclei and, therefore, do likely alter intracellular calcium flux, leading to changes in neuronal responsivity. Exciting new data also point to a potential role for allopregnanolone to influence the activity of autonomic circuits, likely through GABA_A receptor function. Modulation of allopregnanolone within the NTS results in transient hypotension, suggesting a role of neurosteroids in the regulation of blood pressure that could include the maintenance of critical inhibitory signaling within the dorsal vagal complex. However, our understanding of the role of sex steroids in autonomic circuits is still limited, and much more work must be done.

Perspectives and Significance

In summary, this review aims to discuss our understanding of the role sex hormones play in autonomic brain-stem circuits. Sex differences exist in a wide array of the autonomic nervous system’s regulatory function, including the control of cardiovascular, gastrointestinal, and metabolic systems. Although our understanding of mechanism(s) is limited, both estrogen and progesterone play a role in establishing these sex differences, and their exact role in altered neuronal excitability is specific to each autonomic brain region. This review also suggests a novel contributor to the hormone milieu, allopregnanolone. Taken together, significantly more research must be done to elucidate how sex hormones influence autonomic circuities. These studies should continue to include single-hormone replacement but expand to include genetic models and understudied hormones like progesterone and allopregnanolone.

GRANTS

This work was supported by American Heart Association Scientist Development Grant 16SDG26590000 to C. R. Boychuk. This work was also supported by the NIH Jointly Sponsored Predoctoral Training Program in the Neurosciences Training Grant T32 NS082145 (to S. Fedorchak) and NIH-sponsored Cardiovascular Training in Texas Training Grant T32 HL007446 (to E. L. Littlejohn).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.L.L., S.F., and C.R.B. prepared figures; E.L.L., S.F., and C.R.B. drafted manuscript; E.L.L., S.F., and C.R.B. edited and revised manuscript; E.L.L., S.F., and C.R.B. approved final version of manuscript.