The aim of this book

The aim of this book is to introduce students to the language and concepts of neuroendocrinology, including how the neuroendocrine system influences behavior. It began with a consideration of the many chemical messengers in the body, classified as “true” hormones, neurohormones, neurotransmitters, pheromones, parahormones, prohormones, growth factors, cytokines, adipokines, vitamins and neuropeptides. As more became known about the neuroendocrine system, it became clear that these classifications are not unambiguous and a single chemical might fit into two or more classes of messenger and perform different functions. This means that while the classification of chemical messengers is useful to begin the study of neuroendocrinology, by the end it provides little help in understanding the different actions of peptides, steroids, neuropeptides and neurotransmitters on different target cells, even within the same tissue.

The hormones of the endocrine and pituitary glands are generally accepted as the major components of the neuroendocrine system. However, the traditional endocrine function of hormones being released into the bloodstream to act on peripheral target cells represents only a small part of the neuroendocrine activity of hormones such as testosterone, cholecystokinin or somatostatin. These hormones also have significant effects in the brain, via specific receptors, and can alter neural regulation of autonomic reflexes, behavior and emotional states. The hypothalamus provides the link between the brain and the traditional endocrine system and provides the locus for external factors, such as environmental influences, to regulate endocrine target organs. Thus, while the endocrine system consists of a number of closed-loop feedback systems that maintain homeostatic control over the synthesis, storage, release and deactivation of hormones, external stimuli can alter these systems. For example, environmental stimuli, social interactions and cognitive factors can greatly alter the functioning of the endocrine system by altering the neurotransmitter/neuropeptide pathways that regulate the release of hypothalamic hormones.

As illustrated in Figures 6.5 and 6.11, pituitary (LH, FSH and ACTH) and steroid (cortisol) hormone levels in the bloodstream fluctuate dramatically over short periods of time (minutes to hours). In addition, hormones such as GH, ACTH and melatonin show marked circadian variations in their secretion patterns (Figure 6.5). These patterns are physiologically important; for example, we saw in the case of LH secretion, a continuous release, rather than a pulsatile secretion, will not stimulate the ovaries or testes correctly (Figure 7.4). In other words, fertility is dependent on an appropriate pulsatile LH signal reaching the gonads. This principle might be generally applicable to all pituitary hormone secretions. The measurement, or assay, of hormone levels is therefore an important clinical goal, as well as a crucial aid in understanding how hormone levels in blood are regulated and how the neuroendocrine system functions in health and disease. This chapter thus begins with an examination of the methods for measuring hormone levels in the circulation.

Analysis of hormone levels

The level of a circulating hormone can be measured directly in blood samples or estimated by measuring hormone levels in the saliva, urine or feces, measuring urinary metabolites, or by using bioassays. The determination of glucocorticoids levels in hair, for example, is a way to detect long-term exposure to stress.

8.1.1 Direct measurement of circulating hormones

In the past 20 years, there have been striking changes in the analytical techniques used to estimate hormone levels. Until recently, the benchmark in determination of hormone levels was the radioimmunoassay. However, this method, employing antibodies specific to each hormone, and radioactively labeled hormones, is slow, labor-intensive and raised safety problems in the use and disposal of radioactive materials. It is now routine to analyze hormone levels using rapid and automated chemiluminescent or immunometric assays that produce data in a matter of hours, rather than days.

A widely used assay is the Enzyme-linked Immunosorbent Assay (ELISA).

The pituitary gland

The pituitary gland, which is also called the hypophysis, is attached to the hypothalamus at the base of the brain (Figure 3.1). Secretion of the hormones of the pituitary gland is regulated by the hypothalamus and it is through the hypothalamic-pituitary connection that external and internal stimuli can influence the release of the pituitary hormones, thus producing the neural-endocrine interaction. The pituitary has been called the body's “master gland” because its hormonal secretions stimulate a variety of endocrine glands to synthesize and secrete their own hormones. However, it is really the hypothalamus that is the master gland, because it controls the pituitary.

The pituitary gland consists of two primary organs: the anterior pituitary (adenohypophysis or pars distalis) which is a true endocrine gland, and the posterior pituitary (neurohypophysis) which is formed from neural tissue and is an extension of the hypothalamus (Figure 3.1). The pituitary gland is attached to the hypothalamus by the pituitary (hypophyseal) stalk. Further details of the anatomy and physiology of the pituitary gland can be found elsewhere (Norman and Litwack 1997; Amar and Weiss 2003; Boron and Boulpaep 2005; Gardner and Shoback 2011).

The neurohypophysis (posterior pituitary)

The neurohypophysis consists of neural tissue and contains the nerve terminals (about 100,000) of axons whose cell bodies are located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. The axons of these large magnocellular neurosecretory cells project down from the hypothalamus through the part of the pituitary stalk called the infundibulum and terminate in the posterior pituitary gland (Figure 3.2). The neurosecretory cells of the PVN and SON manufacture the hormones oxytocin and vasopressin (also called antidiuretic hormone, ADH), which are transported down the axons and stored in nerve terminals in the posterior pituitary. The axon terminals in the posterior pituitary are surrounded by supporting cells called pituicytes.

In this second edition of An Introduction to Neuroendocrinology, we have rewritten and greatly extended the original content. The revised text includes entirely new reference lists and a complete new set of illustrations. The book reflects the many advances that have occurred in the study of neuroendocrinology during the past 20 years. Nevertheless, and although the text is based largely on modern references, our primary aim is to provide an introductory description of mammalian neuroendocrine control systems. Several books are available that cover this topical and clinically relevant field, but, although valuable, these tend to be advanced texts of the edited, multi-author type. Our book is designed to provide the basic principles necessary to understand how the brain controls, and responds to, the endocrine hormones. It will be suitable for a variety of different students and especially those who might not have been previously exposed to a focused course in neuroendocrinology. Thus, students in psychology, biology and science should be able to master much of the basic material. However, the book is also highly appropriate for honors students and first-year graduate students in physiology, anatomy, neuroscience and medicine. This book is therefore designed for students in two levels of classes: introductory classes, in which all of the material will be new to the student, and more advanced classes, in which the students will be familiar with many of the terms and concepts through courses in biology, physiology, psychology or neuroscience, but who have not studied neuroendocrinology as an integrated discipline.

This book offers an overall outline of the neuroendocrine system and will provide the vocabulary necessary to understand the interaction between hormones and the brain. In addition, we provide a concise description of those topics that must underpin any attempt to learn, and to teach, neuroendocrinology. For example, there are chapters on basic neuroscience (neurotransmitters and neuropeptides), the physiology of the endocrine glands (hormones), receptors and receptor signaling mechanisms (e.g. G proteins; nuclear receptors), hormone assay and gene expression techniques (e.g. ELISA; in situ hybridization) and a description of the immune system, with particular emphasis on the integration of immune and neuroendocrine pathways.

The endocrine glands

The location of the human endocrine glands is shown in Figure 2.1. The pineal gland is a small gland lying deep between the cerebral cortex and the cerebellum at the posterior end of the third ventricle in the middle of the brain. The hypothalamus exerts some degree of control over most of the endocrine glands through the release of neurohormones, neuropeptides and neurotransmitters. The pituitary gland hangs from the bottom of the hypothalamus at the base of the brain and sits in a small cavity of bone above the roof of the mouth.

The thyroid gland is located in the neck and the small parathyroid glands are embedded in the surface of the thyroid. In the chest is the thymus gland, which is very important for the production of T lymphocytes that play a critical role in the immune response. The heart and lungs also act as endocrine glands that secrete hormones. The gastrointestinal (GI) tract, consisting of the stomach and intestines, is also an important source of hormones. The liver secretes several hormones such as somatomedin (also called IGF-1), important for growth. The adrenal glands are complex endocrine glands situated on top of the kidneys. The pancreas secretes hormones involved in regulating blood sugar levels and the kidney also produces hormone-like chemicals. The testes and ovaries produce gonadal hormones, or sex hormones, which, in addition to the maintenance of fertility and sex characteristics, have important effects on behavior. During pregnancy, the placenta acts as an endocrine gland. The endocrine glands occur in similar locations in all vertebrates. A large endocrine gland is fat (adipose tissue) which can be found beneath the skin (subcutaneous), in the abdominal cavity surrounding the heart and GI tract (see Figure 2.1), and within tissues such as liver and muscle. Fat secretes a variety of hormones called adipokines. Finally, two of the largest tissues in the body – skeletal muscle and bone – secrete factors that act in an endocrine fashion.

The endocrine system is driven by hormones released into the bloodstream in order to regulate other, distant organs (see Figure 1.3). These hormones are chemical messages that are decoded by specific recognition sites, or receptors, located in the target cells. Hormones are synthesized and stored in endocrine cells and, when required, they are released into the circulatory system. A number of hormones are transported in the bloodstream by carrier proteins. For example, sex hormone binding globulin is specifically responsible for transport of estradiol and testosterone. Other proteins such as albumin are less specific and serve to transport a variety of hormones. Hormone synthesis, storage, release, transport and deactivation occur through a variety of different mechanisms, depending on the chemical structure of the hormone. For example, peptides such as oxytocin are different in almost every respect from steroid hormones like estradiol. For this reason, the first section of this chapter will examine the chemical structure of hormones.

The chemical structure of hormones

In terms of chemical structure, hormones can be divided into three major groups: (1) steroid hormones; (2) amines; and (3) peptide hormones (Table 7.1). Steroid hormones, like estradiol, are different from the other two groups because of their very low solubility in blood. This is because they are essentially hydrocarbons and therefore need a binding protein to carry them in the bloodstream. A further distinction is the mechanism of their synthesis; steroids and amines are produced from precursors (such as cholesterol and tyrosine, respectively) via specific enzymes, whereas peptides are encoded by specific genes (DNA) that transcribe messenger RNA (mRNA) that is then translated into precursor peptides.

7.1.1 Steroid hormones

The steroid hormones are biosynthesized from cholesterol in the adrenal cortex and gonads. Adrenal steroids include cortisol and aldosterone and the gonadal steroids are progesterone, testosterone and estradiol (see Table 7.1). Note that some steroids are also made in the brain, fat tissue and placenta.

Many chemical messengers regulate neural activity, including neurotransmitters (Chapter 5), steroid hormones (Chapter 9) and peptide hormones (Chapter 10). This chapter, and Chapter 12, examines how the class of chemical messengers termed neuropeptides regulates neural activity. The topic of neuropeptides is now an extensive one and we divide the coverage into two parts. This chapter will describe the classification and synthesis of neuropeptides and their co-localization with classical neurotransmitters. Chapter 12 examines the functions of neuropeptides in the brain and neuroendocrine system.

Classification of neuropeptides

The realization that neuropeptides can act as neurotransmitters took place after the discovery of most of the “classical,” or small molecule, neurotransmitters, such as NE, glutamate and ACh. Perhaps the simplest and obvious difference between these two classes of neurotransmitter is the size of the molecules; neuropeptides range in size from 2 to at least 40 amino acids, whereas a molecule such as NE is derived from a single amino acid (tyrosine) (see Table 7.1). Another difference is that neuropeptides are more versatile in their range of biological activities. For example, some peptide hormones are synthesized in endocrine glands, in fat cells and in the GI tract, but are also produced in the brain, where they act as neurotransmitters or neuromodulators. Another significant distinguishing characteristic of neuropeptides is their mode of synthesis. Classical neurotransmitters such as amino acids or catecholamines are formed by two or three enzymatic steps, often in the nerve terminal. Neuropeptides, on the other hand, are synthesized from large prepropeptides in the neuronal cell body (e.g. see section 7.2.1; Figure 7.2).

In terms of nomenclature, peptide hormones were traditionally and sensibly named after the first function they were known to serve. Thus, the pituitary hormones (ACTH, TSH, FSH, GH, etc.) were named for their actions at their target cells; for example, TSH stimulates the thyroid gland. Hypothalamic-releasing hormones (CRH, TRH, GnRH, GHRH, etc.) were named for their functions at pituitary target cells; and the hormones of the gastrointestinal (GI) tract (CCK, VIP, gastrin, etc.) were named based on their gastrointestinal functions.

In previous chapters, we focused on the neuroendocrine system in terms of a variety of hypothalamic neurotransmitter and hormonal messengers. In Chapter 1, these messengers were seen to act via endocrine, paracrine, autocrine, intracrine and neuroendocrine mechanisms. So far, however, we have not discussed in detail how target cells detect and respond to such messages. Chapter 5 introduced this story by illustrating how neurotransmitters, neurohormones and peptide hormones affect their target cells through receptors localized to the cell membrane. Examples of these receptors are illustrated in Figures 5.2 (ion channel; GABA receptor) and 5.13 (G-protein-coupled receptor) and this type of receptor will be covered in more detail in Chapter 10. The location of receptors on the outside of cells, that is, in the cell membrane, is important for at least two reasons: (1) because peptide hormones are large, water soluble (hydrophilic) molecules which cannot easily pass through the cell membrane; and (2) because cells such as neurons must respond very quickly (seconds) to neurotransmitters like GABA or glutamate that do not have to enter the cell. In marked contrast, steroid hormones (testosterone, estradiol, progesterone, glucocorticoids and mineralocorticoids; Figure 7.3), and thyroid hormones, are small lipophilic (fat soluble) molecules that can readily diffuse through the cell membranes into any cell in the body. As we shall see in this chapter, target cells for steroid and thyroid hormones have receptors that are located inside the cell. These cells therefore respond relatively slowly (minutes to hours) to hormonal stimulation (see Figure 9.1). In brief, the steroid hormone is transported in the blood and released from a binding globulin before freely moving through the cell membrane. Unoccupied steroid hormone receptors (R) are coupled to a molecular chaperone (HSP90; heat shock protein 90) that stabilizes R in the correct shape. When the hormone binds to the receptor-HSP complex, the HSP dissociates and the remaining steroid hormone-receptor complex dimerizes before it enters the cell nucleus. The steroid-R dimer complex then binds to responsive genes via specific hormone response elements (HRE).

As noted in Chapter 11, the human genome contains about 90 genes that encode neuropeptide precursors (pre-propeptides). The biologically active neuropeptide products of the prepropeptides number at least 100, and there are likely to be many more waiting to be discovered. Neuropeptides are synthesized in a wide variety of neurons in many brain regions and more often than not are co-localized and co-released with classical neurotransmitters. Should neuropeptides therefore be categorized as neurotransmitters? An alternative description is that of neuromodulator, since in many instances they modify the neural effects of classical neurotransmitters. A good example of this is shown in Figure 11.2, where co-release of a neuropeptide totally modifies the influence of the co-localized neurotransmitter on a postsynaptic neuron. This chapter will illustrate the neurotransmitter and neuromodulator actions of neuropeptides on the neuroendocrine system, the autonomic nervous system (ANS) and the central nervous system. First, however, we will explore whether neuropeptides are best described as neurotransmitters or neuromodulators, or both.

Neurotransmitter and neuromodulator actions of neuropeptides: a dichotomy or a continuum?

An initial useful exercise is to establish criteria by which a neuromodulator could be defined. Recall that in Chapter 5 several criteria were established to ascertain whether a neurochemical might be considered to be a neurotransmitter (Table 5.1). In brief, these are: (1) synthesized in neurons; (2) present in the presynaptic nerve terminals, usually contained in vesicles, and released into the synapse in amounts sufficient to stimulate the postsynaptic cell; (3) whether endogenously released or applied exogenously, a neurotransmitter should have the same effect on the postsynaptic cell (i.e. it activates the same ion channels or second messenger pathways); (4) receptors specific to the neurotransmitter should be present postsynaptically; (5) receptor antagonists should prevent #3; and (6) a specific deactivating mechanism should exist in the synapse.

Strictly speaking, all of these criteria apply equally well to neuropeptides, with some qualifications. For example, classical neurotransmitters are made by enzymatic transformation (criterion #1) of a single amino acid transported into the neuron from the circulation, whereas neuropeptides are produced via changes in gene expression.

Chapters 2 and 3 surveyed the hormones of the endocrine and pituitary glands. This chapter outlines the functions of the hypothalamus, and the hypothalamic neurosecretory cells and examines the role of the hypothalamus in controlling the release of pituitary hormones.

Functions of the hypothalamus

The hypothalamus is located at the base of the forebrain, below the thalamus (see Figure 3.1), and is divided into two halves, along the midline, by the third ventricle, which is filled with cerebrospinal fluid (CSF). As shown in a coronal (frontal) section in Figure 4.1, the hypothalamus contains many groups, or nuclei, of nerve cell bodies. The medial basal hypothalamus, consisting of the VMH, ARC and median eminence, is often referred to as the “endocrine hypothalamus” because of its neuroendocrine functions. For students interested in further details, a description of the anatomy of the hypothalamus can be found elsewhere (Norris 2007; Page 2006; Squire et al. 2008).

It is beyond the scope of this book to consider in detail the many and complex roles of the hypothalamus in maintaining normal bodily functions. But bear in mind that this brain center exerts an amazing diversity of critical controls, including growth, reproduction, temperature control, metabolism and body weight, emotional behavior (anger, fear, euphoria), motivational arousal (hunger, thirst, aggression and sexual arousal), circadian rhythms, stress and fluid balance. It contains multiple internal connections between neurons, but in addition receives neural information from other brain regions such as amygdala, hippocampus and spinal cord. The hypothalamus is well supplied with blood vessels and is therefore the recipient of essential information from the bloodstream, such as temperature and hormone levels. Thus, we can appreciate the importance of the hypothalamus in integrating and responding to all of this information by modifying its output of neural and neuroendocrine signaling.

These functions of the hypothalamus can be “localized” to particular nuclei, although any boundaries, such as those outlined in Figure 4.1, should be regarded only as approximate guides.

Hormones, the brain and behavior

Neuroendocrinology is the study of how the brain controls the endocrine systems that keep us alive and able to reproduce. However, an essential and critical characteristic of this neural control of the endocrine systems is that endocrine hormones in turn have profound effects on brain function through feedback systems. Research on hormones and the brain is intensive and covers many fields: from cell and molecular biology and genetics to anatomy, physiology, pharmacology, biochemistry, medicine, psychiatry and psychology. This book will examine the interactions between hormones, the brain and behavior. Thus, the primary focus will be on how the endocrine and nervous systems affect each other to produce an integrated functional neuroendocrine system that influences physiological and behavioral responses. As preliminary background reading, students are referred to any modern text on Human Physiology (see “Further reading” at the end of this chapter).

When you hear the term “hormone,” for example steroid hormone, you think of the endocrine glands and how their secretions influence physiological responses in the body, but this is only part of the picture. Many of the endocrine glands (although not all of them) are influenced by the pituitary gland, the so-called “master gland,” and the pituitary is itself controlled by various hormones secreted from the hypothalamus, a part of the brain situated directly above the pituitary gland. The release of hypothalamic hormones is in turn regulated by neurotransmitters released from nerve cells (neurons) in the brain. Some neurotransmitters released within the brain also control behavior, and the secretion of neurotransmitters from specific nerve cells can be modulated by the level of specific endocrine hormones in the circulation. This is called hormone feedback. Thus, neurotransmitter release influences both hormones and behavior and, in turn, hormones regulate the release of neurotransmitters. This interaction between hormones, the brain and behavior involves a wide variety of chemical messengers which are described in this chapter.

Cytokines were introduced in Chapter 1 (section 1.4.9) as signaling molecules secreted by cells of the immune system. They are important components of the interconnected neural, endocrine and immune systems (see Figure 1.2). For example, various types of stress, including academic examinations, influence the immune system via an activation of the hypothalamic-pituitary-adrenal axis and the secretion of glucocorticoids (see Figures 6.1 and 6.4). Likewise, an immune response that stimulates white blood cells to produce cytokines, for example following an infection, has effects on the hypothalamic control of various hormones such as ACTH, GH and PRL. Since most immune cells have receptors for these hormones, the immune system is affected directly by pituitary hormones as well as by adrenal output of glucocorticoids or catecholamines (Figure 1.2). The immune system, therefore, via secretion of cytokines, participates in a classic neuroendocrine feedback system. There are also profound sex differences in immune responses and some of this variation is due to the effects of sex steroids, such as testosterone and estradiol. For example, 80 percent of patients with autoimmune diseases (e.g. rheumatoid arthritis; multiple sclerosis) are women; 60 percent of adult asthma cases are women; and men are at least 1.6-fold more likely than women to die from malignant cancers (Klein 2012).

This chapter begins with an overview of those cells of the immune system that secrete cytokines, and then discusses the immune functions of the thymus gland and its hormones. The roles of cytokines in the immune response to antigens (i.e. substances that cause an immune response) and in the development of blood cells are then summarized and the neuromodulatory effects of cytokines on the brain and neuroendocrine system are examined. This is followed by a discussion of the neural and endocrine regulation of the immune system and the hypothalamic integration of neural, endocrine and immune systems.

The cells of the immune system

The immune system consists of many different cell types, including several that secrete cytokines; i.e. monocytes, macrophages, T lymphocytes (T cells), B lymphocytes (B cells) and natural killer (NK) cells (see Figure 13.1).

Behavioral neuroendocrinology involves the study of the interactive effects of steroid and peptide hormones, neuropeptides, cytokines and neurotransmitters on behavior. Previous chapters have mentioned the role of hypothalamic nuclei in behavior (section 4.1), the behavioral effects of neurotransmitter agonists and antagonists (section 5.8.4), the neuroendocrine correlates of psychiatric disorders (section 6.8), the behavioral functions of steroid hormones (sections 9.9.3 and 9.9.4), the cognitive and behavioral effects of neuropeptides (section 12.7) and the effects of cytokines on the brain and behavior (section 13.5). The present chapter discusses behavioral methods used in the study of neuroendocrinology, the neural and genetic mechanisms mediating the effects of hormones on behavior, and some of the special problems involved in conducting behavioral neuroendocrinology research.

Neuroendocrine research utilizes several specific methods such as immunoradiometric assays for quantifying hormone levels (Chapter 8), immunohistochemistry (Chapter 9) and immuno fluorescence techniques for localizing hormones (Chapters 11 and 12). The study of behavioral neuroendocrinology relies on specific behavioral methodologies or behavioral bioassays. As discussed in Chapter 8 (section 8.1.3), a bioassay measures physiological changes in an animal or cell culture to determine the concentration or potency of a hormone in the circulation. Thus, for example, the size of a cock's comb is a bioassay for blood testosterone level and in rats the size and weight of the adrenal glands is a bioassay for the level of circulating ACTH or corticosterone. A behavioral bioassay measures behavioral changes to estimate the concentration or potency of a hormone.

Behavioral bioassays

A behavioral bioassay requires precise qualitative (verbal) descriptions of the behaviors of interest and accurate quantitative (mathematical) measures of the latency, frequency and duration of these behaviors. Thus, the measurement of behavior involves two stages: the observation and description of units of behavior and the quantitative measurement of these behavior units. Before these procedures can begin, however, one must determine which behaviors to record.