Eric S. Wohleb
Proper immune function is critical to maintain homeostasis, recognize and eliminate pathogens, and promote tissue repair. Primary and secondary immune organs receive input from the autonomic nervous system and immune cells express receptors for epinephrine, norepinephrine, and/or acetylcholine. Through direct signaling the autonomic nervous system controls immune function by altering immune cell development, initiating redistribution of immune cells throughout the body, and promoting molecular pathways that shift immune cell reactivity. This neuroimmune communication allows the autonomic nervous system to shape immune function based on physiological and psychological demands.
Penile erection is a part of the human male sexual response, involving desire, excitation (erection), orgasm (ejaculation), and resolution, and autonomic nerves are involved in all phases. Autonomic innervation of smooth-muscle cells of the erectile tissue is provided by the cavernous nerve. Motor and sensory innervation is derived from the pudendal nerves and their terminal branches, that is, the dorsal nerves of the penis, which carry impulses from receptors harbored in the penile skin, prepuce, and glans. Erection begins with an increased flow in the pudendal arteries and dilatation of the cavernous arteries and helicine arterioles in association with relaxation of the smooth muscles of the trabecular network, causing engorgement of blood in the corpora. This leads to compression of subtunical venules by the resistant tunica albuginea and erection. During detumescence these events are reversed.
Paul J. May, Anton Reiner, and Paul D. Gamlin
The functions of the eye are regulated by and dependent upon the autonomic nervous system. The parasympathetic nervous system controls constriction of the iris and accommodation of the lens via a pathway with preganglionic motor neurons in the Edinger-Westphal nucleus and postganglionic motor neurons in the ciliary ganglion. The parasympathetic nervous system regulates choroidal blood flow and the production of aqueous humor through a pathway with preganglionic motor neurons in the superior salivatory nucleus and postganglionic motor neurons in the pterygopalatine (sphenopalatine) ganglion. The sympathetic nervous system controls dilation of the iris and may modulate the outflow of aqueous humor from the eye. The sympathetic preganglionic motor neurons lie in the intermediolateral cell column at the first level of the thoracic cord, and the postganglionic motor neurons are found in the superior cervical ganglion.
The central pathways controlling different autonomic functions in the eye are found in a variety of locations within the central nervous system. The reflex response of the iris to changes in luminance levels begins with melanopsin-containing retinal ganglion cells in the retina that project to the olivary pretectal nucleus. This nucleus then projects upon the Edinger-Westphal preganglionic motoneurons. The dark response that produces maximal pupillary dilation involves the sympathetic pathways to the iris. Pupil size is also regulated by many other factors, but the pathways to the parasympathetic and sympathetic preganglionic motoneurons that underlie this are not well understood. Lens accommodation is controlled by premotor neurons located in the supraoculomotor area. These also regulate the pupil, and control vergence angle by modulating the activity of medial rectus, and presumably lateral rectus, motoneurons. Pathways from the frontal eye fields and cerebellum help regulate their activity. Blood flow in the choroid is regulated with respect to systemic blood pressure through pathways through the nucleus of the tractus solitarius. It is also regulated with respect to luminance levels, which likely involves the suprachiasmatic nucleus, which receives inputs from melanopsin-containing retinal ganglion cells, and other areas of the hypothalamus that project upon the parasympathetic preganglionic neurons of the superior salivatory nucleus that mediate choroidal vasodilation.
Thad E. Wilson and Kristen Metzler-Wilson
Thermoregulation is a key physiologic homeostatic process and is subdivided into autonomic, behavioral, and adaptive divisions. Autonomic thermoregulation is a neural process related to the sympathetic and parasympathetic nervous systems. Autonomic thermoregulation is controlled at the subcortical level to alter physiologic processes of heat production and loss to maintain internal temperature. Mammalian, including human, autonomic responses to acute heat or cold stresses are dependent on environmental conditions and species genotype and phenotype, but many similarities exist. Responses to an acute heat stress begin with the sensation of heat, leading to central processing of the information and sympathetic responses via end organs, which can include sweat glands, vasculature, and airway and cardiac tissues. Responses to an acute cold stress begin with the sensation of cold, which leads to central processing of the information and sympathetic responses via end organs, which can include skeletal and piloerector muscles, brown adipose tissue, vasculature, and cardiac tissue. These autonomic responses allow homeostasis of internal temperature to be maintained across a wide range of external temperatures for most mammals, including humans. At times, uncompensable thermal challenges occur that can be maintained for only limited periods of time before leading to pathophysiologic states of hyperthermia or hypothermia.
Natalia Duque-Wilckens and Brian C. Trainor
Aggressive behavior plays an essential role in survival and reproduction across animal species—it has been observed in insects, fish, reptiles, and mammals including humans. Even though specific aggressive behaviors are quite heterogeneous across species, many of the underlying mechanisms modulating aggression are highly conserved. For example, in a variety of species arginine vasopressin (AVP) and its homologue vasotocin in the hypothalamus, play an important role in regulating aggressive behaviorssuch as territorial and inter male aggression. Similarly in the medial amygdala, activation of a subpopulation of GABAergic neurons promotes aggression, while the prefrontal cortex exerts inhibitory control over aggressive behaviors. An important caveat in the aggression literature is that it is focused primarily on males, probably because in most species males are more aggressive than females. However, female aggression is also highly prevalent in many contexts, as it can affect access to resources such as mates, food, and offspring survival. Although it is likely that many underlying mechanisms are shared between sexes, there is sex specific variation in aggression, type, magnitude, and contexts, which suggests that there are important sex differences in how aggression is regulated. For example, while AVP acts to modulate aggression in both male and female hamsters, it increases male aggression but decreases female aggression. These differences can occur at the extent of neurotransmitter or hormones release, sensitivity (i.e., receptor expression), and/or molecular responses.
In response to changes in metabolic demand, the cardiovascular and respiratory systems are regulated in a highly coordinated fashion, such that both ventilation and cardiac output increase in a parallel fashion, thus maintaining a relatively constant level of arterial blood PO2, PCO2, and pH. In addition, external alerting stimuli that trigger defensive or orienting behavioral responses also trigger coordinated cardiorespiratory changes that are appropriate for the particular behavior. Furthermore, environmental challenges such as hypoxia or submersion evoke complex cardiovascular and respiratory response that have the effect of increasing oxygen uptake and/or conserving the available oxygen.
The brain mechanisms that are responsible for generating coordinated cardiorespiratory responses can be divided into reflex mechanisms and feedforward (central command) mechanisms. Reflexes that regulate cardiorespiratory function arise from a wide variety of internal receptors, and include those that signal changes in blood pressure, the level of blood oxygenation, respiratory activity, and metabolic activity. In most cases more than one reflex is activated, so that the ultimate cardiorespiratory response depends upon the interaction between different reflexes. The essential central pathways that subserve these reflexes are largely located within the brainstem and spinal cord, although they can be powerfully modulated by descending inputs arising from higher levels of the brain. The brain defense mechanisms that regulate the cardiorespiratory responses to external threatening stimuli (e.g., the sight, sound, or odor of a predator) are highly complex, and include both subcortical and cortical systems. The subcortical system, which includes the basal ganglia and midbrain colliculi as essential components, is phylogenetically ancient and generates immediate coordinated cardiorespiratory and motor responses to external stimuli. In contrast, the defense system that includes the cortex, hypothalamus, and limbic system evolved at a later time, and is better adapted to generating coordinated responses to external stimuli that involve cognitive appraisal.
Eliot A. Brenowitz
Animals produce communication signals to attract mates and deter rivals during their breeding season. The coincidence in timing results from the modulation of signaling behavior and neural activity by sex steroid hormones associated with reproduction. Adrenal steroids can influence signaling for aggressive interactions outside the breeding season. Androgenic and estrogenic hormones act on brain circuits that regulate the motivation to produce and respond to signals, the motor production of signals, and the sensory perception of signals. Signal perception, in turn, can stimulate gonadal development.
Jeremy C. Borniger and Luis de Lecea
The hypocretins (also known as orexins) are selectively expressed in a subset of lateral hypothalamic neurons. Since the reports of their discovery in 1998, they have been intensely investigated in relation to their role in sleep/wake transitions, feeding, reward, drug abuse, and motivated behavior. This research has cemented their role as a subcortical relay optimized to tune arousal in response to various salient stimuli. This article reviews their discovery, physiological modulation, circuitry, and integrative functionality contributing to vigilance state transitions and stability. Specific emphasis is placed on humoral and neural inputs regulating hcrt neural function and new evidence for an autoimmune basis of the sleep disorder narcolepsy. Future directions for this field involve dissection of the heterogeneity of this neural population using single-cell transcriptomics, optogenetic, and chemogenetics, as well as monitoring population and single cell activity. Computational models of the hypocretin network, using the “flip-flop” or “integrator neuron” frameworks, provide a fundamental understanding of how this neural population influences brain-wide activity and behavior.
Brian P. Kenealy and Ei Terasawa
Female reproduction is an interplay between the hypothalamus, pituitary, and ovaries. While the gonadotropin releasing hormone (GnRH) neuron in the hypothalamus regulates gonadal function through the pituitary, GnRH neuronal activity is also profoundly influenced by ovarian steroid hormones. GnRH is released from GnRH neurons in a pulsatile manner after integration of a diverse array of internal and external milieus. Since the discovery of the mammalian GnRH molecule, over a dozen GnRH forms have been identified in the animal kingdom, and large numbers of publications in various lab animal and human studies suggest that GnRH neurons are regulated by multiple neuromodulators in the brain, such as kisspeptin, neurokinin B, β-dynorphin, neuropeptide Y, GnIH, GABA, glutamate, and glial factors. A recent emerging concept is that steroids synthesized locally in the hypothalamus, namely, neuroestradiol and neuroprogesterone, also contribute to the regulation of GnRH neuronal activity, and hence female reproduction. Together with modulation by various inputs and ovarian steroid feedback, GnRH neurons are responsible for puberty, cyclic ovulation, and menopause.
Margaret M. McCarthy
Sex differences in the brain are established by the differential gonadal steroid hormonal milieu experienced by developing male and female fetuses and newborns. Androgen production by the testis starts in males prior to birth resulting in a brief developmental window during which the brain is exposed to high levels of steroid. Androgens and aromatized estrogens program the developing brain toward masculinized physiology and behavior that is then manifest in adulthood. In rodents, the perinatal programming of sex-specific adult mating behavior provides a model system for exploring the mechanistic origins of brain sex differences.
Microglia are resident in the brain and provide innate immunity. Previously considered restricted to response to injury, these cells are now thought to be major contributors to the sculpting of developing neural circuits. This role extends to being an important component of the sexual differentiation process and has opened the door for exploration into myriad other aspects of neuroimmunity and inflammation as possible determinants of sex differences. In humans, males are at greater risk for more frequent and/or more severe neuropsychiatric and neurological disorders of development, many of which include prenatal inflammation as an additional risk factor. Emerging clinical and preclinical evidence suggests male brains experience a higher inflammatory tone early in development, and this may have its origins in the maternal immune system. Collectively, these disparate observations coalesce into a coherent picture in which brain development diverges in males versus females due to a combination of gonadal steroid action and neuroinflammation, and the latter increases the risk to males of developmental disorders.
Jill E. Schneider
During the evolution of animals, survival and reproduction depended upon mechanisms that maintained internal homeostasis in the face of environmental change. These environmental changes included fluctuations in ambient temperature, food availability, humidity, day length, and population density. Most, if not all, of these variables have effects on the availability of energy, and most vertebrate species have mechanisms that sense energy availability and adjust behavioral priorities accordingly. For example, when the availability of food and potential mating partners is stable and abundant, brain mechanisms often inhibit ingestive behavior, increase energy expenditure, and give priority to courtship and mating. In response to severe energy shortages, brain mechanisms are likely to stimulate foraging, food hoarding, and overeating. These same deficits often delay reproductive development or inhibit adult reproductive behavior. Such adaptations involve the integration of sensory signals with peripheral hormone signals and central effectors, and they are key to understanding health and disease, particularly obesity, eating disorders, and diabetes.
The link between energy balance and reproduction recurs repeatedly, whether in the context of the sensory-somatic system, the autonomic nervous system, or the neuroendocrine cascades. Peripheral signals that are detected by receptors on vagal and splanchnic nerves are relayed to the caudal hindbrain. This brain area contains the effectors for peripheral hormone secretion and for chewing and swallowing, and this same brain area contains receptors for humoral and metabolic signals from peripheral circulation. The caudal hindbrain is therefore a strong candidate for integration of multiple signals that control the initiation of meals, meal size, energy storage, and energy expenditure, including the energy expended on reproduction. There are some differences between the reproductive and ingestive mechanisms, but there are also many striking similarities. There are still gaps in our knowledge about the nature and location of metabolic receptors and the pathways to their effectors. Some of the most promising research is designed to shed light on how hormonal signals might be enhanced or modulated by the peripheral energetic condition (e.g., the level of oxidizable metabolic fuels).
Danielle S. Stolzenberg, Kimberly L. Hernandez-D'Anna, Oliver J. Bosch, and Joseph S. Lonstein
For female mammals, caring for young until weaning or even longer is an extension of the reproductive burden that begins at insemination. Given the high price females potentially pay for failing to transmit genetic material to future generations, a multitude of interacting endocrine, neuroendocrine, and other neurochemical determinants are in place to ensure competent maternal caregiving by the time the offspring are born. Achieving this high maternal competency at parturition seems effortless but is quite a feat given that many nulliparous and parentally inexperienced female mammals are more prone to ignore, if not outright harm, conspecific neonates. There are important roles for ovarian steroids (e.g., estradiol and progesterone), adrenal steroids (e.g., glucocorticoids), and neuropeptide hormones (e.g., prolactin, oxytocin, arginine-vasopressin, and corticotropin-releasing factor) released during pregnancy, parturition, and postpartum in the onset and maintenance of caregiving behaviors in a broad range of commonly studied animals including rats, mice, rabbits, sheep, and primates. It is especially remarkable that the same collection of hormones influences caregiving similarly across these diverse animals, although to varying degrees. In addition to the well-known effects of hormones and neuropeptides on motherhood, more recent research indicates that experience-dependent epigenetic effects are also powerful modulators of the same neural substrates that can influence maternal responding.
Paul E. Micevych and Melinda A. Mittelman-Smith
In the last two decades of the 20th century, key findings in the field of estrogen signaling completely changed our understanding of hormones: first, steroidogenesis was demonstrated in the CNS; second, a vast majority of cells in the nervous system were shown to have estrogen receptors; third, a second nuclear estrogen receptor (ERß) was cloned; and finally, “nuclear” receptors were shown to be present and functional in the cell membrane. Shortly thereafter, even more membrane estrogen receptors were discovered. Steroids (estrogens, in particular) began to be considered as neurotransmitters and their receptors were tethered to G protein-coupled receptor signaling cascades. In some parts of the brain, levels of steroids appeared to be independent of those found in the circulation and yet, circulating steroids had profound actions on the brain physiology. In this review, we discuss the interaction of peripheral and central estrogen action in the context of female reproduction—one of the best-studied aspects of steroid action. In addition to reviewing the evidence for steroidogenesis in the hypothalamus, we review membrane-localized nuclear receptors coupling to G protein-signaling cascades and the downstream physiological consequences for reproduction. We will also introduce newer work that demonstrates cell signaling for a common splice variant of estrogen receptor-α (ERα), and membrane action of neuroprogesterone in regulating estrogen positive feedback.
Mineralocorticoid Receptors and Glucocorticoid Receptors in HPA Stress Responses During Coping and Adaptation
Edo Ronald de Kloet and Marian Joëls
The glucocorticoid hormones cortisol and corticosterone coordinate circadian events and are master regulators of the stress response. These actions of the glucocorticoids are mediated by mineralocorticoid receptors (NR3C2, or MRs) and glucocorticoid receptors (NR3C1, or GRs). MRs bind the natural glucocorticoids cortisol and corticosterone with a 10-fold higher affinity than GRs. The glucocorticoids are inactivated only in the nucleus tractus solitarii (NTS), rendering the NTS-localized MRs aldosterone-selective and involved in regulation of salt appetite. Everywhere else in the brain MRs are glucocorticoid-preferring. MR and GR are transcription factors involved in gene regulation but recently were also found to mediate rapid non-genomic actions. Genomic MRs, with a predominant localization in limbic circuits, are important for the threshold and sensitivity of the stress response system. Non-genomic MRs promote appraisal processes, memory retrieval, and selection of coping style. Activation of GRs makes energy substrates available and dampens initial defense reactions. In the brain, GR activation enhances appetitive- and fear-motivated behavior and promotes memory storage of the selected coping style in preparation of the future. Thus, MRs and GRs complement each other in glucocorticoid control of the initiation and termination of the stress response, suggesting that the balance in MR- and GR-mediated actions is crucial for homeostasis and health.
Circadian rhythm is the approximately 24-hour rhythmicity that regulates physiology and behavior in a variety of organisms. The mammalian circadian system is organized in a hierarchical manner. Molecular circadian oscillations driven by genetic feedback loops are found in individual cells, whereas circadian rhythms in different systems of the body are orchestrated by the master clock in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. SCN receives photic input from retina and synchronizes endogenous rhythms with the external light/dark cycles. SCN regulates circadian rhythms in the peripheral oscillators via neural and humoral signals, which account for daily fluctuations of the physiological processes in these organs. Disruption of circadian rhythms can cause health problems and circadian dysfunction has been linked to many human diseases.
D. Grahame Hardie and A. Mark Evans
AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that monitors the levels of AMP and ADP relative to ATP. If increases in AMP:ATP and/or ADP:ATP ratios are detected (indicating a reduction in cellular energy status), AMPK is activated by the canonical mechanism involving both allosteric activation and enhanced net phosphorylation at Thr172 on the catalytic subunit. Once activated, AMPK phosphorylates dozens of downstream targets, thus switching on catabolic pathways that generate ATP and switching off anabolic pathways and other energy-consuming processes. AMPK can also be activated by non-canonical mechanisms, triggered either by glucose starvation by a mechanism independent of changes in adenine nucleotides, or by increases in intracellular in response to hormones, mediated by the alternate upstream kinase CaMKK2.
AMPK is expressed in almost all eukaryotic cells, including neurons, as heterotrimeric complexes comprising a catalytic α subunit and regulatory β and γ subunits. The α subunits contain the kinase domain and regulatory regions that interact with the other two subunits. The β subunits contain a domain that, with the small lobe of the kinase domain on the α subunit, forms the “ADaM” site that binds synthetic drugs that are potent allosteric activators of AMPK, while the γ subunits contain the binding sites for the classical regulatory nucleotides, AMP, ADP, and ATP.
Although much undoubtedly remains to be discovered about the roles of AMPK in the nervous system, emerging evidence has confirmed the proposal that, in addition to its universal functions in regulating energy balance at the cellular level, AMPK also has cell- and circuit-specific roles at the whole-body level, particularly in energy homeostasis. These roles are mediated by phosphorylation of neural-specific targets such as ion channels, distinct from the targets by which AMPK regulates general, cell-autonomous energy balance. Examples of these cell- and circuit-specific functions discussed in this review include roles in the hypothalamus in balancing energy intake (feeding) and energy expenditure (thermogenesis), and its role in the brainstem, where it supports the hypoxic ventilatory response (breathing), increasing the supply of oxygen to the tissues during systemic hypoxia.
Jonathan M. Beckel and William C. de Groat
Functions of the lower urinary tract to store and periodically eliminate urine are regulated by a complex neural control system in the brain and lumbosacral spinal cord that coordinates the activity of smooth and striated muscles of the bladder and urethral outlet via a combination of voluntary and reflex mechanisms. Many neural circuits controlling the lower urinary tract exhibit switch-like patterns of activity that turn on and off in an all-or-none manner. During urine storage, spinal sympathetic and somatic reflexes are active to maintain a quiescent bladder and a closed outlet. During micturition, these spinal storage reflexes are suppressed by input from the brain, while parasympathetic pathways in the brain are activated to produce a bladder contraction and relaxation of the urethra. The major component of the micturition switching circuit is a spinobulbospinal parasympathetic pathway that consists of essential relay circuitry in the periaqueductal gray and pontine micturition center. These circuits in the rostral brain stem are, in turn, regulated by inputs from the forebrain that mediate voluntary control of micturition. Thus neural control of micturition is organized as a hierarchical system in which spinal storage reflexes and supraspinal voiding reflexes are regulated voluntarily by higher centers in the brain. In young children the voluntary mechanisms are undeveloped and voiding is purely reflex. Voluntary control emerges during maturation of the nervous system and depends on learned behavior. Diseases or injuries of the nervous system in adults cause re-emergence of involuntary micturition, leading to urinary incontinence.
Ruth I. Wood and Kathryn G. Wallin-Miller
Anabolic-androgenic steroids (AAS) are both performance-enhancing substances and drugs of abuse. Although AAS are banned in competitive sports, they are widely used by both elite and rank-and-file athletes. All AAS are derived from testosterone, the principle endogenous androgen produced by the testes of adult men. While AAS increase muscular strength and athletic performance, they also have serious consequences for health and behavior. AAS are implicated in maladaptive behavioral and cognitive changes such as increased risk-taking and altered decision-making. However, effects of AAS on cognition are not well understood. Studies of human AAS users are limited by an inability to control for pre-existing psychopathology and behavioral differences. Furthermore, in order to understand AAS effects on behavior, it is important to discover how AAS impact the brain. Animal models of AAS abuse parallel human studies to uncover effects on cognition, decision-making, and underlying neurobiological mechanisms. In operant discounting tests, rats treated with chronic high-dose testosterone are less sensitive to effort, punishment, and delay but are more sensitive to uncertainty. Likewise, they demonstrate impaired cognitive flexibility when tested for set-shifting and reversal learning. It appears that AAS induce many of these cognitive changes via effects on the mesocorticolimbic dopamine system, particularly through the dopamine D1- and D2-like receptors in subnuclei of the nucleus accumbens. AAS also have rewarding effects mediated by similar neural circuits. In preclinical studies, animals will voluntarily self-administer AAS. Human users may develop dependence. These findings highlight the vulnerability of brain circuits controlling cognition and reward to androgens at high doses.
Divine C. Nwafor, Allison L. Brichacek, Sreeparna Chakraborty, Catheryne A. Gambill, Stanley A. Benkovic, and Candice M. Brown
The blood-brain barrier (BBB) is a dynamic structural interface between the brain and periphery that plays a critical function in maintaining cerebral homeostasis. Over the past two decades, technological advances have improved our understanding of the neuroimmune and neuroendocrine mechanisms that regulate a healthy BBB. The combination of biological sex, sex steroids, age, coupled with innate and adaptive immune components orchestrates the crosstalk between the BBB and the periphery. Likewise, the BBB also serves as a nexus within the hypothalamic-pituitary-adrenal (HPA) and gut-brain-microbiota axes. Compromised BBB integrity permits the entry of bioactive molecules, immune cells, microbes, and other components that migrate into the brain parenchyma and compromise neuronal function. A paramount understanding of the mechanisms that determine the bidirectional crosstalk between the BBB and immune and endocrine pathways has become increasingly important for implementation of therapeutic strategies to treat a number of neurological disorders that are significantly impacted by the BBB. Examples of these disorders include multiple sclerosis, Alzheimer’s disease, stroke, epilepsy, and traumatic brain injury.
Ashlyn Swift-Gallant and S. Marc Breedlove
While prenatal sex hormones guide the development of sex-typical reproductive structures, they also act on the developing brain, resulting in sex differences in brain and behavior in animal models. Stemming from this literature is the prominent hypothesis that prenatal neuroendocrine factors underlie sex differences in human sexual orientation, to explain why most males have a preference for female sexual partners (gynephilia), whereas most females display a preference for male sexual partners (androphilia). Convergent evidence from experiments of nature and indirect markers of prenatal hormones strongly support a role for prenatal androgens in same-same sexual orientations in women, although this finding is specific to a subset of lesbians who are also gender nonconforming (“butch”). More gender-conforming lesbians (“femmes”) do not show evidence of increased prenatal androgens. The literature has been more mixed for male sexual orientation: some report evidence of low prenatal androgen exposure, while others report evidence of high androgen levels and many other studies find no support for a role of prenatal androgen exposure in the development of androphilia in males. Recent evidence suggests there may be subgroups of gay men who owe their sexual orientation to distinct biodevelopmental mechanisms, which could account for these mixed findings. Although this research is young, it is similar to findings from lesbian populations, because gay men who are more gender nonconforming, and report a preference for receptive anal sex, differ on markers of prenatal development from gay men who are more gender conforming and report a preference for insertive anal sex. This chapter concludes with future research avenues including assessing whether multiple biodevelopmental pathways underlie sexual orientation and whether neuroendocrine factors and other biological mechanisms (e.g., immunology, genetics) interact to promote a same-sex sexual orientation.