Sex diversity in the 21st century: Concepts, frameworks, and approaches for the future of neuroendocrinology.

Sex is ubiquitous and variable throughout the animal kingdom. Historically, scientists have used reductionist methodologies that rely on a priori sex categorizations, in which two discrete sexes are inextricably linked with gamete type. However, this binarized operationalization does not adequately reflect the diversity of sex observed in nature. This is due, in part, to the fact that sex exists across many levels of biological analysis, including genetic, molecular, cellular, morphological, behavioral, and population levels. Furthermore, the biological mechanisms governing sex are embedded in complex networks that dynamically interact with other systems. To produce the most accurate and scientifically rigorous work examining sex in neuroendocrinology and to capture the full range of sex variability and diversity present in animal systems, we must critically assess the frameworks, experimental designs, and analytical methods used in our research. In this perspective piece, we first propose a new conceptual framework to guide the integrative study of sex. Then, we provide practical guidance on research approaches for studying sex-associated variables, including factors to consider in study design, selection of model organisms, experimental methodologies, and statistical analyses. We invite fellow scientists to conscientiously apply these modernized approaches to advance our biological understanding of sex and to encourage academically and socially responsible outcomes of our work. By expanding our conceptual frameworks and methodological approaches to the study of sex, we will gain insight into the unique ways that sex exists across levels of biological organization to produce the vast array of variability and diversity observed in nature.

An updated field guide for snark hunting: Comparative contributions to behavioral neuroendocrinology in the era of model organisms.

Studying neuroendocrine behavioral regulatory mechanisms in a variety of species across vertebrate groups is critical for determining how they work in natural contexts, how they evolved, and ultimately what can be generalized from them, potentially even to humans. All of the above are difficult, at best, if work within our field is exclusively done in traditional laboratory organisms. The importance of comparative approaches for understanding the relationships between hormones and behavior has been recognized and advocated for since our field's inception through a series of papers centered upon a poetic metaphor of Snarks and Boojums, all of which have articulated the benefits that come from studying a diverse range of species and the risks associated with a narrow focus on "model organisms." This mini-review follows in the footsteps of those powerful arguments, highlighting some of the comparative work since the latest interactions of the metaphor that has shaped how we think about three major conceptual frameworks within our field, two of them formalized - the Organization/Activation Model of sexual differentiation and the Social Brain Network - and one, context-dependency, that is generally associated with virtually all modern understandings of how hormones affect behavior. Comparative approaches are broadly defined as those in which the study of mechanism is placed within natural and/or evolutionary contexts, whether they directly compare different species or not. Studies are discussed in relation to how they have either extended or challenged generalities associated with the frameworks, how they have shaped subsequent work in model organisms to further elucidate neuroendocrine behavioral regulatory mechanisms, and how they have stimulated work to determine if and when similar mechanisms influence behavior in our own species.

How technical progress reshaped behavioral neuroendocrinology during the last 50 years and some methodological remarks.

The first issue of Hormones and Behavior was published 50 years ago in 1969, a time when most of the techniques we currently use in Behavioral Endocrinology were not available. Researchers have during the last 5 decades developed techniques that allow measuring hormones in small volumes of biological samples, identify the sites where steroids act in the brain to activate sexual behavior, characterize and quantify gene expression correlated with behavior expression, modify this expression in a specific manner, and manipulate the activity of selected neuronal populations by chemogenetic and optogenetic techniques. This technical progress has considerably transformed the field and has been very beneficial for our understanding of the endocrine controls of behavior in general, but it did also come with some caveats. The facilitation of scientific investigations came with some relaxation of methodological exigency. Some critical controls are no longer performed on a regular basis and complex techniques supplied as ready to use kits are implemented without precise knowledge of their limitations. We present here a selective review of the most important of these new techniques, their potential problems and how they changed our view of the hormonal control of behavior. Fortunately, the scientific endeavor is a self-correcting process. The problems have been identified and corrections have been proposed. The next decades will obviously be filled with exciting discoveries in behavioral neuroendocrinology.

Mini-review: Epigenetic mechanisms that promote transgenerational actions of endocrine disrupting chemicals: Applications to behavioral neuroendocrinology.

It is our hope this mini-review will stimulate discussion and new research. Here we briefly examine the literature on transgenerational actions of endocrine disrupting chemicals (EDCs) on brain and behavior and their underlying epigenetic mechanisms including: DNA methylation, histone modifications, and non-coding RNAs. We stress that epigenetic modifications need to be examined in a synergistic manner, as they act together in situ on chromatin to change transcription. Next we highlight recent work from one of our laboratories (VGC). The data provide new evidence that the sperm genome is poised for transcription. In developing sperm, gene enhancers and promoters are accessible for transcription and these activating motifs are also found in preimplantation embryos. Thus, DNA modifications associated with transcription factors during fertilization, in primordial germ cells (PGCs), and/or during germ cell maturation may be passed to offspring. We discuss the implications of this model to EDC exposures and speculate on whether natural variation in hormone levels during fertilization and PGC migration may impart transgenerational effects on brain and behavior. Lastly we discuss how this mechanism could apply to neural sexual differentiation.

On the Neuroendocrinopathy of Critical Illness. Perspectives for Feeding and Novel Treatments.

Typical for critical illnesses are substantial alterations within the hypothalamic-anterior pituitary-peripheral hormonal axes that are proportionate to the risk of poor outcome. These neuroendocrine responses to critical illness follow a biphasic pattern. The acute phase (first hours to days) is characterized by an increased release of anterior pituitary hormones, whereas altered target-organ sensitivity and hormone metabolism result in low levels of the anabolic peripheral effector hormones and contribute to the substantially elevated levels of the catabolic hormone cortisol. The prolonged phase of critical illness is hallmarked by a uniform suppression of the neuroendocrine axes, predominantly of central/hypothalamic origin, which contributes to the low (or insufficiently high in the case of cortisol) circulating levels of the target-organ hormones. Several of the acute-phase adaptations to critical illness are due to or accentuated by the concomitant fasting. Accepting the lack of macronutrients as well as the neuroendocrine responses to such fasting in the acute phase of critical illness has shown to beneficially affect outcome. In contrast, the neuroendocrine alterations that occur in the chronic phase of illness while patients are fully fed contribute to bone and skeletal muscle wasting and impose risk of adrenocortical atrophy. The combined administration of those hypothalamic releasing factors, which have been identified as suppressed or deficient during prolonged critical illness, may be a promising strategy to enhance recovery. The potential impact of treatment with such hypothalamic releasing factors on recovery from critical illness as well as on long-term rehabilitation should be investigated in future randomized controlled clinical trials.

Neuroendocrine control of the onset of puberty.

This chapter is based on the Geoffrey Harris Memorial Lecture presented at the 8th International Congress of Neuroendocrinology, which was held in Sydney, August 2014. It provides the development of our understanding of the neuroendocrine control of puberty since Harris proposed in his 1955 monograph (Harris, 1955) that "a major factor responsible for puberty is an increased rate of release of pituitary gonadotrophin" and posited "that a neural (hypothalamic) stimulus, via the hypophysial portal vessels, may be involved." Emphasis is placed on the neurobiological mechanisms governing puberty in highly evolved primates, although an attempt is made to reverse translate a model for the timing of puberty in man and monkey to non-primate species.

Neuropeptidergic regulation of reproduction in insects.

Successful animal reproduction depends on multiple physiological and behavioral processes that take place in a timely and orderly manner in both mating partners. It is not only necessary that all relevant processes are well coordinated, they also need to be adjusted to external factors of abiotic and biotic nature (e.g. population density, mating partner availability). Therefore, it is not surprising that several hormonal factors play a crucial role in the regulation of animal reproductive physiology. In insects (the largest class of animals on planet Earth), lipophilic hormones, such as ecdysteroids and juvenile hormones, as well as several neuropeptides take part in this complex regulation. While some peptides can affect reproduction via an indirect action (e.g. by influencing secretion of juvenile hormone), others exert their regulatory activity by directly targeting the reproductive system. In addition to insect peptides with proven activities, several others were suggested to also play a role in the regulation of reproductive physiology. Because of the long evolutionary history of many insect orders, it is not always clear to what extent functional data obtained in a given species can be extrapolated to other insect taxa. In this paper, we will review the current knowledge concerning the neuropeptidergic regulation of insect reproduction and situate it in a more general physiological context.

Translational Neuroendocrinology of Human Skin: Concepts and Perspectives.

Human skin responds to numerous neurohormones, neuropeptides, and neurotransmitters that reach it via the vasculature or skin nerves, and/or are generated intracutaneously, thus acting in a para- and autocrine manner. This review focuses on how neurohormones impact on human skin physiology and pathology. We highlight basic concepts, major open questions, and translational research perspectives in cutaneous neuroendocrinology and argue that greater emphasis on neuroendocrine human skin research will foster the development of novel dermatological therapies. Furthermore, human skin and its appendages can be used as highly accessible and clinically relevant model systems for probing nonclassical, ancestral neurohormone functions. This calls for close interdisciplinary collaboration between dermatologists, skin biologists, neuroendocrinologists, and neuropharmacologists.

Neuroendocrinology and its quantitative development: a bioengineering view.

Biomedical engineering is clearly present in modern neuroendocrinology, and indeed has come to embrace it in many respects. First, we briefly review the origins of endocrinology until neuroendocrinology, after a long saga, was established in the 1950's decade with quantified results made possible by the radioimmunoassay technique (RIA), a development contributed by the physical sciences. However, instrumentation was only one face of the quantification process, for mathematical models aiding in the study of negative feedback loops, first rather shyly and now at a growing rate, became means building the edifice of mathematical neuroendocrinology while computer assisted techniques help unravel the associated genetic aspects or the nature itself of endocrine bursts by numerical deconvolution analysis. To end the note, attention is called to the pleiotropic characteristics of neuroendocrinology, which keeps branching off almost endlessly as bioengineering does too.

Diving into the brain: deep-brain imaging techniques in conscious animals.

In most species, survival relies on the hypothalamic control of endocrine axes that regulate critical functions such as reproduction, growth, and metabolism. For decades, the complexity and inaccessibility of the hypothalamic-pituitary axis has prevented researchers from elucidating the relationship between the activity of endocrine hypothalamic neurons and pituitary hormone secretion. Indeed, the study of central control of endocrine function has been largely dominated by 'traditional' techniques that consist of studying in vitro or ex vivo isolated cell types without taking into account the complexity of regulatory mechanisms at the level of the brain, pituitary and periphery. Nowadays, by exploiting modern neuronal transfection and imaging techniques, it is possible to study hypothalamic neuron activity in situ, in real time, and in conscious animals. Deep-brain imaging of calcium activity can be performed through gradient-index lenses that are chronically implanted and offer a 'window into the brain' to image multiple neurons at single-cell resolution. With this review, we aim to highlight deep-brain imaging techniques that enable the study of neuroendocrine neurons in awake animals whilst maintaining the integrity of regulatory loops between the brain, pituitary and peripheral glands. Furthermore, to assist researchers in setting up these techniques, we discuss the equipment required and include a practical step-by-step guide to performing these deep-brain imaging studies.

The organizational-activational hypothesis as the foundation for a unified theory of sexual differentiation of all mammalian tissues.

The 1959 publication of the paper by Phoenix et al. was a major turning point in the study of sexual differentiation of the brain. That study showed that sex differences in behavior, and by extension in the brain, were permanently sexually differentiated by testosterone, a testicular secretion, during an early critical period of development. The study placed the brain together in a class with other major sexually dimorphic tissues (external genitalia and genital tracts), and proposed an integrated hormonal theory of sexual differentiation for all of these non-gonadal tissues. Since 1959, the organizational-activational theory has been amended but survives as a central concept that explains many sex differences in phenotype, in diverse tissues and at all levels of analysis from the molecular to the behavioral. In the last two decades, however, sex differences have been found that are not explained by such gonadal hormonal effects, but rather because of the primary action of genes encoded on the sex chromosomes. To integrate the classic organizational and activational effects with the more recently discovered sex chromosome effects, we propose a unified theory of sexual differentiation that applies to all mammalian tissues.

How and when to use dried blood spots in psychoneuroendocrinological research.

OBJECTIVE: The term "dried blood spot" (DBS) refers to a sampling technique in which capillary whole blood is spotted on filter paper. Given the possibility to determine a wide range of hormones and related biomarkers in DBS, the method should be of interest to researchers in psychoneuroendocrinology. So far, however, the how and when of using DBS in this context have not been outlined. METHODS: A review of the literature was conducted in order to describe the materials and procedures necessary to determine relevant biological markers from DBS (how to use DBS). In addition, a comparison of the DBS method with other sampling techniques was undertaken and examples of its previous use in psychoneuroendocrinology were provided (when to use DBS). RESULTS: Both dyadic and DBS self-sampling are feasible, and a number of protocols are available to determine endocrine and immune, genetic and epigenetic markers. Decisions to use DBS instead of venous blood or saliva sampling should mainly be guided by whether it is sensible and feasible to determine the parameter of interest in whole blood obtained from DBS. In addition, DBS are well suited for large study populations with specific vulnerabilities, and for remotely located studies with budgetary constraints. CONCLUSION: Dried blood spots are a promising material as well as a simple sampling technique for psychoneuroendocrinological research. Future efforts should be directed at continuing to adapt existing serum and plasma analysis protocols for use with DBS, and at testing the feasibility of DBS self-sampling in field studies.

From organotypic culture to body-on-a-chip: A neuroendocrine perspective.

The methods used to study neuroendocrinology have been as diverse as the discoveries to come out of the field. Maintaining live neurones outside of a body in vitro was important from the beginning, building on methods that dated back to at least the first decade of the 20th Century. Neurosecretion defines an essential foundation of neuroendocrinology based on work that began in the 1920s and 1930s. Throughout the first half of the 20th Century, many paradigms arose for studying everything from single neurones to whole organs in vitro. Two of these survived as preeminent systems for use throughout the second half of the century: cell cultures and explant systems. Slice cultures and explants that emerged as organotypic technologies included such neuroendocrine organs such as the brain, pituitary, adrenals and intestine. The vast majority of these studies were carried out in static cultures for which media were changed over a time scale of days. Tissues were used for experimental techniques such as electrical recording of neuronal physiology in single cells and observation by live microscopy. When maintained in vitro, many of these systems only partially capture the in vivo physiology of the organ system of interest, often because of a lack of cellular diversity (eg, neuronal cultures lacking glia). Modern microfluidic methodologies show promise for organ systems, ranging from the reproductive to the gastrointestinal to the brain. Moving forward and striving to understand the mechanisms that drive neuroendocrine signalling centrally and peripherally, there will always be a need to consider the heterogeneous cellular compositions of organs in vivo.

Thirty years of neuroendocrinology: Technological advances pave the way for molecular discovery.

Since the 1950s, the systems level interactions between the hypothalamus, pituitary and end organs such as the adrenal, thyroid and gonads have been well known; however, it is only over the last three decades that advances in molecular biology and information technology have provided a tremendous expansion of knowledge at the molecular level. Neuroendocrinology has benefitted from developments in molecular genetics, epigenetics and epigenomics, and most recently optogenetics and pharmacogenetics. This has enabled a new understanding of gene regulation, transcription, translation and post-translational regulation, which should help direct the development of drugs to treat neuroendocrine-related diseases.

Conceptual endophenotypes: A strategy to advance the impact of psychoneuroendocrinology in precision medicine.

Psychobiological research has generated a tremendous amount of findings on the psychological, neuroendocrine, molecular and environmental processes that are directly relevant for mental and physical health, but have overwhelmed our capacity to meaningfully absorb, integrate, and utilize this knowledge base. Here, we reflect about suitable strategies to improve the translational success of psychoneuroendocrinological research in the era of precision medicine. Following a strategy advocated by the National Research Council and the tradition of endophenotype-based research, we advance here a new approach, termed "conceptual endophenotypes". We define the contextual and formal criteria of conceptual endophenotypes, outline criteria for filtering and selecting information, and describe how conceptual endophenotypes can be validated and implemented at the bedside. As proof-of-concept, we describe some of our findings from research that has adopted this approach in the context of stress-related disorders. We argue that conceptual endophenotypes engineer a bridge between the bench and the bedside. This approach readily lends itself to being continuously developed and implemented. Recent methodological advances, including digital phenotyping, machine learning, grassroots collaboration, and a learning healthcare system, may accelerate the development and implementation of this conceptual endophenotype approach.

Minireview: The neuroendocrinology of the suprachiasmatic nucleus as a conductor of body time in mammals.

Circadian rhythms in physiology and behavior are regulated by a master clock resident in the suprachiasmatic nucleus (SCN) of the hypothalamus, and dysfunctions in the circadian system can lead to serious health effects. This paper reviews the organization of the SCN as the brain clock, how it regulates gonadal hormone secretion, and how androgens modulate aspects of circadian behavior known to be regulated by the SCN. We show that androgen receptors are restricted to a core SCN region that receives photic input as well as afferents from arousal systems in the brain. We suggest that androgens modulate circadian behavior directly via actions on the SCN and that both androgens and estrogens modulate circadian rhythms through an indirect route, by affecting overall activity and arousal levels. Thus, this system has multiple levels of regulation; the SCN regulates circadian rhythms in gonadal hormone secretion, and hormones feed back to influence SCN functions.

Effective use of microarrays in neuroendocrine research.

The development of microarray technology makes it possible to simultaneously assay the expression level of hundreds to tens of thousands of mRNA transcripts in one experiment. Genome-wide transcriptional analysis has increasing importance for many areas of neuroendocrinology research. The expense and technical complexity of microarray experiments can make it difficult to navigate the terrain of rival platforms and technologies. In this review, we provide a practical view and comparison of various microarray technologies. Affymetrix arrays, high-density cDNA arrays, membrane arrays and experimental design and data analysis are all discussed by researchers currently using these techniques to study gene regulation in neuroendocrine tissues.

NeuroScholar's electronic laboratory notebook and its application to neuroendocrinology.

Scientists continually relate information from the published literature to their current research. The challenge of this essential and time-consuming activity increases as the body of scientific literature continues to grow. In an attempt to lessen the challenge, we have developed an Electronic Laboratory Notebook (ELN) application. Our ELN functions as a component of another application we have developed, an open-source knowledge management system for the neuroscientific literature called NeuroScholar (http://www. neuroscholar. org/). Scanned notebook pages, images, and data files are entered into the ELN, where they can be annotated, organized, and linked to similarly annotated excerpts from the published literature within Neuroscholar. Associations between these knowledge constructs are created within a dynamic node-and-edge user interface. To produce an interactive, adaptable knowledge base. We demonstrate the ELN's utility by using it to organize data and literature related to our studies of the neuroendocrine hypothalamic paraventricular nucleus (PVH). We also discuss how the ELN could be applied to model other neuroendocrine systems; as an example we look at the role of PVH stressor-responsive neurons in the context of their involvement in the suppression of reproductive function. We present this application to the community as open-source software and invite contributions to its development.

Exploring the "brain-skin connection": Leads and lessons from the hair follicle.

Research into how the central nervous system (CNS) and the skin of mammals are physiologically connected and how this "brain-skin connection" may be therapeutically targeted in clinical medicine has witnessed a renaissance. A key element in this development has been the discovery that mammalian skin and its appendages, namely human scalp hair follicles (HFs), not only are important, long-underestimated target tissues for classical neurohormones, neurotrophins and neuropeptides, but also are eminent peripheral tissue sources for the production and/or release of these neuromediators. This essay summarizes the many different levels of biology at which human scalp HFs respond to and generate a striking variety of neurohormones, and portrays HFs as prototypic, cyclically remodelled miniorgans that utilize these neurohormones to autoregulate their growth, hair shaft production, rhythmic organ transformation, pigmentation, mitochondrial energy metabolism, and immune status. The essay also explores how preclinical research on human scalp HFs can be exploited to unveil and explore "novel" and clinically as yet untapped, but most likely ancestral functions of neurohormones within mammalian epithelial biology that still impact substantially on human skin physiology. Arguably, systematic investigation of the "brain-skin connection" is one of the most intriguing current research frontiers in investigative dermatology, not the least since it has reversed the traditional CNS focus in studying the interactions between two key organ systems by placing the skin epithelium on center stage.