58: Sexual Differentiation of the Nervous System

• Genes and Hormones Determine Physical Differences Between Males and Females

  • Chromosomal Sex Directs the Gonadal Differentiation of the Embryo

  • Gonads Synthesize Hormones That Promote Sexual Differentiation

  • Steroid Hormones Act by Binding to Specific Receptors

• Sexual Differentiation of the Nervous System Generates Sexually Dimorphic Behaviors

  • A Sexually Dimorphic Neural Circuit Controls Erectile Function

  • A Sexually Dimorphic Neural Circuit Controls Song Production in Birds

  • A Sexually Dimorphic Neural Circuit in the Hypothalamus Controls Mating Behavior

• Environmental Cues Control Some Sexually Dimorphic Behaviors

  • Pheromones Control Partner Choice in Mice

  • Early Experience Modifies Later Maternal Behavior

• Sexual Dimorphism in the Human Brain May Correlate with Gender Identity and Sexual Orientation

• An Overall View

Few words are more loaded with meaning than the word "sex." Sexual activity is a biological imperative and a major human preoccupation. The physical differences between men and women that underlie partner recognition and reproduction are obvious to all of us, and their developmental origins are well understood. In contrast, our understanding of behavioral differences between the sexes is primitive. In many cases their very existence remains controversial, and the origins of those that have been clearly demonstrated remain unclear.

In this chapter we first briefly summarize the embryological basis of sexual differentiation. We then discuss at greater length the behavioral differences between the two sexes, focusing on those differences or dimorphisms for which some neurobiological basis has been found. These dimorphisms include physiological responses (erection, lactation), drives (maternal behavior), and even more complex behaviors (gender identity). In analyzing these dimorphisms we will discuss three issues.

First, what are the genetic origins of sexual differences? Human males and females have a complement of 23 chromosomal pairs, and only one differs between the sexes. Females have a pair of X chromosomes (and are therefore "XX"), whereas males have one copy of the X chromosome paired with a Y chromosome (XY). The other 22 chromosome pairs, called autosomes, are shared between males and females. We will see that some genetic determinants arise from the presence of a Y chromosome, while others arise from sex-specific patterns of autosomal gene expression that exert their impact during development.

Second, how are differences in genes and gene expression translated into differences between the brains of men and women? We will see that key intermediates are the sex hormones, a set of steroids that includes testosterone and estrogens. These hormones act during embryogenesis as well as postnatally, first organizing the physical development of both genitalia and brain regions, and later activating particular physiological and behavioral responses. Hormonal regulation is especially complex because the nervous system, which is profoundly influenced by sex steroids, also controls their synthesis. This feedback loop may help to explain how the external environment, including social and cultural factors, can ultimately shape sexual dimorphism at a neural level.

Third, what are the crucial neural differences that underlie sexually dimorphic behaviors? Clear physical and molecular differences between the brains of men and women have been found. These differences imply that neural circuitry differs between the sexes, and in a few cases these distinctions in connectivity are directly related to behavioral differences. In other cases, however, sexually dimorphic behaviors appear to result from differential usage of the same basic circuits.

Before proceeding we must define the usage of two words that are commonly confused with each other: sex and gender. As a descriptor of biological differences between men and women, the word sex is used in three ways. First, anatomical sex refers to overt differences including the differences in the external genitalia as well as other sexual characteristics such as the distribution of body hair. Gonadal sex refers to the presence of male or female gonads, the testes or ovaries. Finally, chromosomal sex refers to the distribution of the sex chromosomes between females (XX) and males (XY).

Whereas sex is a biological term, gender encompasses the collection of social behaviors and mental states that typically differ between males and females. Gender role is the set of behaviors and social mannerisms that is typically distributed in a sexually dimorphic fashion within the population. Toy preferences in children as well as distinctive attire are some examples of gender roles that can distinguish males from females. Gender identity is the feeling of belonging to the category of the male or female sex. Importantly, gender identity is distinct from sexual orientation, the erotic responsiveness displayed toward members of one or the other sex.

Are gender and sexual orientation genetically determined? Or are they social constructs molded by cultural expectations and personal experience? As the examples in this chapter will illustrate, we are still far from untangling the contributions of genes and environment to such complex phenomena. However, our recognition that genes and experience interact to shape neural circuits gives us a more realistic framework with which to answer this question compared to our predecessors, who were constrained by the simplistic view that genes and experience acted in mutually exclusive ways.

Chromosomal Sex Directs the Gonadal Differentiation of the Embryo

Sex determination is the embryonic process whereby chromosomal sex directs the differentiation of the gonadal sex of the animal. Surprisingly, this process differs in fundamental ways within the animal kingdom and even among vertebrates. In most mammals, including humans, however, an XY genotype drives differentiation of the embryonic gonad into testes, whereas an XX genotype leads to ovarian differentiation. Hormones produced by the testes and ovaries subsequently direct sexual differentiation of the nervous system and the rest of the body.

It is the presence of the Y chromosome rather than the lack of a second X chromosome that is the crucial determinant of male differentiation. This was first evident in rare individuals born with two or even three X chromosomes in addition to a Y chromosome (XXY or XXXY). These individuals are men who exhibit male-typical traits. In fact, female cells do not have two active X chromosomes. Early in embryogenesis one of the two X chromosomes in each female cell is chosen at random for inactivation and the genes on it are rendered transcriptionally silent. Thus both male and female cells have a single active X chromosome and male cells also have a Y chromosome.

The sex-determining activity of the Y chromosome is encoded by the gene SRY (sex-determining region on Y) whose activity is required for masculinization of the embryonic gonads (Figure 58–1). Inactivation or deletion of SRY leads to complete sex reversal: Individuals are chromosomally male (XY) but externally indistinguishable from females. Conversely, in rare instances SRY translocates to another chromosome (to the X chromosome or an autosome) during spermatogenesis. Such sperm can fertilize eggs to produce individuals who are chromosomally female (XX) but externally male. However, such XX sex-reversed men are infertile, as many of the genes required for sperm function are located on the Y chromosome.

How does SRY instruct the undifferentiated gonads to develop into testes? The female differentiation program appears to be the default mode; patterning genes prime the body and gonads to develop along female-specific pathways. The SRY gene encodes a transcription factor that induces expression of genes, some of which prevent execution of the default program and initiate the process of male gonadal differentiation. One of the best-studied targets of the SRY transcription factor is another transcription factor, SOX9, which is required for differentiation of the testes. SOX9 in turn activates a variety of genes required for formation of testicular Sertoli cells. Thus SRY initiates a cascade of inductive interactions that ultimately lead to male-specific gonad development.

Gonads Synthesize Hormones That Promote Sexual Differentiation

The chromosomal complement of the embryo directs sexual differentiation of the gonads and in turn the gonads determine the sex-specific features of the nervous system and the rest of the body. They do this by secreting hormones. Gonadal hormones have two major roles. Their developmental role is traditionally referred to as organizational because the early effects of hormones on the brain and the rest of the body lead to major, generally irreversible, aspects of cell and tissue differentiation. Later some of the same hormones trigger physiological or behavioral responses. These influences, generally termed activational, are reversible.

One example of an organizational role of gonadal hormones is seen in the differentiation of structures that connect the gonads to the external genitalia. In males the Wolffian duct gives rise to the vas deferens, the seminal vesicles, and the epididymis. In females the Müllerian duct differentiates into the oviduct, the uterus, and the vagina (Figure 58–2). Initially both female (XX) and male (XY) embryos possess Wolffian and Müllerian ducts. In males the developing testes secrete a protein hormone, the Müllerian inhibiting substance (MIS), and a steroid hormone, testosterone. MIS leads to a regression of the Müllerian duct and testosterone induces the Wolffian duct to differentiate into its mature derivatives. In females the absence of MIS permits the Müllerian duct to differentiate into its adult derivatives, and the absence of circulating testosterone causes the Wolffian duct to resorb. Thus the Y chromosome overrides a female default program to generate male gonads, which in turn secrete hormones that override a female default program of genital differentiation.

The action of MIS is largely confined to embryos, but steroid hormones exert effects throughout life—that is, they have activational as well as organizational roles. All of the steroid hormones derive from cholesterol (Figure 58–3). The sex steroids can be divided into androgens, which generally promote male characteristics, and the estrogens plus progesterone that promote female characteristics. The testes produce mostly the androgen testosterone, while the ovaries produce mostly progesterone and an estrogen, 17-β-estradiol. The menstrual cycle is a good example of the activational function of estrogen and progesterone.

A glance at the metabolic relationships among steroid hormones (Figure 58–3) reveals a surprise. The female hormone progesterone is the precursor of the male hormone testosterone, and testosterone is the direct precursor of the female hormone 17-β-estradiol. Thus the enzymes that convert one hormone to the other control not only the level of the hormone but also the "sign" (male or female) of the hormonal effect. Aromatase, the enzyme that converts testosterone to estradiol, is present at high levels in the ovaries but not in the testes. Differential expression of aromatase is the reason for sexual dimorphism in circulating testosterone and estrogen. Aromatase is also expressed in various regions of the brain (Figure 58–4A), and many of the effects of testosterone on neurons are thought to occur after its conversion to estrogen. Testosterone is also converted by the enzyme 5α-reductase into another androgenic steroid, 5α-dihydrotestosterone (DHT), in various target tissues, including the external genitalia. In these tissues DHT is responsible for induction of secondary male characteristics such as facial and body hair and growth of the prostate. Later in life DHT is the culprit in male pattern baldness.

As one can imagine, mutations in genes encoding enzymes involved in steroid hormone biosynthesis have far-reaching consequences. The phenotypes dramatically illustrate both the organizational and activational effects of steroid hormones, as well as the difficulty of neatly distinguishing the two. Here we describe three disorders (Table 58–1).

The first, congenital adrenal hyperplasia or CAH, is a genetic deficiency in the synthesis of corticosteroids by the adrenal glands that results in overproduction of testosterone and related androgens. This condition is autosomal recessive and occurs once in 10,000–15,000 live births. In girls born with CAH, excess androgens lead to some masculinization of the external genitalia, a process called virilization. Virilization clearly reflects the organizational roles of steroids. This condition can be diagnosed at birth and resolved by surgical intervention. Treatment with corticosteroids reduces testosterone levels, permitting these females to undergo puberty and to be fertile.

A second genetic disorder, 5α-reductase II deficiency, can also affect sexual differentiation (Table 58–1). In male fetuses 5α-reductase II is expressed at high levels in the precursor of the external genitalia, where it converts circulating testosterone into DHT. The high local concentrations of DHT virilize the external genitalia. Clinical 5α-reductase II deficiency is inherited in an autosomal recessive manner, and males present at birth with ambiguous (under-virilized) or overtly feminized external genitalia. In many instances, therefore, chromosomally male patients (XY) with this condition are mistakenly raised as females until puberty, at which time the large increase in circulating testosterone virilizes the body hair, musculature, and, most dramatically, the external genitalia.

Steroid Hormones Act by Binding to Specific Receptors

The critical role of steroid receptors in controlling sexual differentiation is well illustrated by patients with a third disorder, complete androgen insensitivity syndrome or CAIS (Table 58–1). Testosterone, estrogen, and progesterone are hydrophobic molecules that are able to diffuse across cell membranes, enter the bloodstream, enter cells in many organs, and bind to intra-cellular ligand-specific receptors. The receptors for these hormones are encoded by distinct but homologous genes.

A single gene encodes a receptor that binds the androgens testosterone and DHT. The androgen receptor binds DHT approximately threefold more tightly than testosterone, accounting for the greater potency of DHT. There is also a single receptor for progesterone (progesterone receptor), whereas two genes encode receptors that bind estrogens (estrogen receptors α and β). The estrogen receptors are present in many tissues of the body, including the brain (Figure 58–4B).

These receptor proteins are transcription factors that bind specific sites in the genome and modulate transcription of target genes. They contain several signature motifs, including a hormone-binding domain, a DNA-binding domain, and a domain that modulates the transcriptional activity of target genes (Figure 58–5A). Hormones activate the transcriptional activity by binding to the receptor. In the absence of ligand the receptors bind to protein complexes that sequester them in the cytoplasm. Upon binding of ligand the receptors dissociate from the complex and enter the nucleus, where they dimerize and bind to specific sequence elements in the promoter and enhancer regions of target genes, modulating their transcription (Figure 58–5B).

Patients with CAIS are chromosomally XY but carry a loss-of-function allele of the X-linked androgen receptor that abolishes cellular responses to testosterone and DHT. Because the pathway of sex determination via SRY remains functional, these patients have testes. However, because of androgen signaling, the Wolffian ducts do not develop, the testes fail to descend, and the external genitalia are feminized. In adulthood most of these patients opt for surgical removal of the testes and hormonal supplementation appropriate for females.

Sex-specific behaviors occur because the nervous system differs between males and females. These differences arise from a combination of genetic factors, such as components of the sex determination pathways, as well as environmental factors, such as social experience. In many cases both genetic and environmental inputs act through the steroid hormone system to sculpt the nervous system. Many anatomical instances of sexual dimorphism have been documented, including differences in the numbers and size of neurons in particular structures as well as differences in the pattern and number of synapses.

It is challenging to trace the chain of causality from environmental or genetic factors to the development of neural dimorphisms and to link these differences to sex-specific behaviors. In this section we examine a few cases in which studies in experimental animals have provided insights. In later sections we ask whether similar mechanisms underlie sexually dimorphic behaviors in humans.

However, before proceeding we note that the ways in which chromosomal mechanisms of sex determination are linked to the cellular processes of sexual differentiation in the central nervous system vary widely among species. In insects sex differences in behavior are independent of hormonal secretion from the gonads, and instead rely exclusively on a sex determination pathway within individual neurons. This mode of sexual differentiation of the brain and behavior is particularly well understood in the fruit fly, where it has been demonstrated that the sex determination cascade initiates expression of a single transcription factor, fruitless, that specifies the entire repertory of male sexual behaviors (Box 58–1).

A Sexually Dimorphic Neural Circuit Controls Erectile Function

The lumbar spinal cord of many mammals, including humans, contains a sexually dimorphic motor center, the spinal nucleus of the bulbocavernosus (SNB). Motor neurons in the SNB innervate the bulbocavernosus muscle, which plays an important part in penile reflexes in males and vaginal movements in females.

In adult rats the male SNB contains many more motor neurons than the female SNB. In addition, male SNB motor neurons are larger in size and have larger dendritic arbors, with a corresponding increase in the number of synapses they receive. Like the SNB motor neurons, the bulbocavernosus muscle is larger in males than females; it is completely absent in the females of some mammalian species. SNB motor neurons also innervate the levator ani muscle, which is involved in copulatory behavior and is also larger in males than females.

How do these differences arise? Initially the circuit is not sexually dimorphic. At birth male and female rats have similar numbers of neurons in the SNB and similar numbers of fibers in the bulbocavernosus and levator ani muscles. In females, however, many motor neurons in the SNB and many fibers in the bulbocavernosus and levator ani muscles die in early postnatal life. Thus this sexual dimorphism arises not by male-specific generation of cells but rather by female-specific cell death.

Perinatal injections of testosterone or DHT can rescue a significant number of the dying neurons and muscle fibers in the female rat. Conversely, treatment of male pups with an androgen receptor antagonist increases the number of dying neurons and muscle fibers. So at a deeper level we see that the dimorphism results from male-specific preservation of motor neurons and muscle fibers that would die in the absence of hormone.

Where does testosterone act to establish this structural dimorphism? Is it primarily a survival factor for the motor neurons, with muscle fibers dying secondarily because they lose their innervation? Or does testosterone act on muscles, which then provide a trophic factor to support the survival of SNB motor neurons? This issue has been examined in rats carrying a mutation of the androgen receptor (tfm allele) that reduces binding of ligand to 10% of normal. The receptor resides on the X chromosome, so all males that carry a mutant gene on their one and only X chromosome are feminized and sterile. For female heterozygotes, the situation is more complicated. As described earlier, one of the X chromosomes is randomly inactivated in each XX female.

Female heterozygotes are therefore mosaics: some cells express a functional androgen receptor allele, others the mutated allele. Each muscle fiber has many nuclei, so most bulbocavernosus muscle fibers in the heterozygous female express functional androgen receptors. Motor neurons have a single nucleus, however, so each neuron is either normal or receptor-deficient. If androgen receptors were required in the neuron, one would expect only receptor-expressing SNB motor neurons to survive, whereas if receptors were required only in muscles, one would expect surviving motor neurons to be a mixture of wild type and mutant.

In fact, the latter situation occurs, indicating that survival of SNB motor neurons does not depend on a neuron-autonomous function of the androgen receptor. Rather, these neurons receive a trophic cue from the androgen-dependent bulbocavernosus and the levator ani muscles (Figure 58–7A). These cues may include the ciliary neurotrophic factor (CNTF) or a related molecule, because mutant male mice lacking a CNTF receptor exhibit a decreased number of SNB motor neurons, typical of females.

Male and female SNB motor neurons also differ in size. Androgens determine the differences in number and size of these neurons in different ways. Studies of tfm mutants showed that androgens exert an organizational effect during early postnatal life through a direct effect on muscle. Low levels of androgens during this critical period lead to an irreversible reduction in the number of SNB motor neurons. Later, androgens act directly on SNB motor neurons to increase the extent of their dendritic arbors. A loss of circulating testosterone, such as that occurring after castration, leads to a dramatic pruning of dendritic arbors; injection of supplemental testosterone to a castrated male rat can restore this dendritic branching pattern (Figure 58–7B). This effect persists in adulthood and is reversible, so it can be viewed as an activational influence. Thus androgens can exert diverse effects, even on a single neuronal type.

A Sexually Dimorphic Neural Circuit Controls Song Production in Birds

Several species of songbirds learn species-specific vocalizations that are used for courtship rituals and territorial marking (see Chapter 60). A set of interconnected brain nuclei controls the learning and production of birdsong (Figure 58–8A). In some songbird species both sexes sing and the structure of the song circuit is similar in males and females. In other species, such as zebra finches and canaries, males alone sing. In these species several song-related nuclei are significantly larger in the male than in the female.

The development of sexual dimorphism in song circuitry has been studied in detail in the zebra finch. In the adult male zebra finch the robust nucleus of the archistriatum (RA) contains fivefold more neurons than does the same nucleus in females. In addition, the afferent projections to RA exhibit a striking sexual dimorphism—only in males does the RA receive input from high vocal centers (HVCs) (Figure 58–8B). These sex differences in cell number and connectivity of RA are not evident until after hatching, when in females a large number of RA neurons die and in males the axons of HVC neurons enter the RA nucleus.

These sexually dimorphic anatomical features are regulated by steroid hormones. When females are supplied with estrogen (or an aromatizable androgen such as testosterone) after hatching, the number of neurons in the RA and the termination pattern in the nucleus are similar to that of the male. However, early hormone administration to young females is not sufficient to masculinize the song nuclei to a size comparable to that of adult males, nor is it sufficient to induce singing in females. To achieve these functions, female birds that receive testosterone or estradiol after hatching must also receive testosterone or dihydrotestosterone (but not estrogen) as adults. Thus steroids play both organizational and activational roles in this system.

A Sexually Dimorphic Neural Circuit in the Hypothalamus Controls Mating Behavior

In many mammalian species the preoptic region of the hypothalamus and a reciprocally connected region, the bed nucleus of the stria terminalis (BNST), play important roles in sexually dimorphic mating behaviors. In male rodents and monkeys these areas are activated during mating behavior; surgical lesions that ablate the preoptic region or the BNST result in deficits in male sexual behavior in male rodents, and also disinhibit female-type sexual receptivity. Thus this region contains neurons that activate and inhibit female sexual behavior. Surgical lesioning of the preoptic hypothalamic region activates male mating routines and inhibits female sexual receptivity in rodents.

Both the preoptic hypothalamus and the BNST are sexually dimorphic, containing more neurons in males compared to females. The sexually dimorphic nucleus of the preoptic area (SDN-POA) also contains significantly more neurons in the male. A male-specific perinatal surge of testosterone promotes survival of neurons in the SDN-POA, whereas in females these same cells gradually die off in the early postnatal period. This development is similar to that in the sexually dimorphic nuclei of the rodent spinal cord and the songbird brain, suggesting that androgen control is a common mechanism for production of sex differences in the size of neuronal populations.

Curiously, the ability of brain testosterone to promote the survival of neurons is likely to be exerted via aromatization into estrogen and subsequent activation of the estrogen receptors (see Figure 58–5). How, then, is the neonatal female brain shielded from the effects of circulating estrogen? In newborn females there is very little estrogen in the circulation, and the small amount present is sequestered by binding to α-fetoprotein, a serum protein. This explains why female mice lacking α-fetoprotein exhibit male-specific behaviors and reduced female-typical sexual receptivity. In this case, then, structural sexual dimorphism does not result from differential effects of androgens and estrogens, but rather from sex differences in the level of hormone available to the target tissue.

Sex-specific behaviors are usually initiated in response to sensory cues in the environment. There are many such cues, and distinct sensory modalities are used in a species-specific manner to elicit a similar response. Courtship rituals can be triggered by species-specific vocalizations, visual signals, odors, and even, in the case of weakly electric fish, by electric discharges. Recent genetic and molecular studies have led to significant insight into how sensory experience controls some of these behaviors in rodents. Here we discuss two examples: the regulation of partner choice by pheromones and the regulation of maternal behavior by experience during infancy.

Pheromones Control Partner Choice in Mice

Many animals rely on their sense of smell to move about, obtain food, and avoid predators. They also rely on pheromones—chemicals that are produced by an animal to affect the behavior of another member of the species. In rodents pheromones can trigger many sexually dimorphic behaviors, including mate choice and aggression (the propensity of males to fight over territory or mates and the tendency of nursing females to attack nest intruders).

Pheromones are detected by neurons in two distinct sensory tissues in the vertebrate nose: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) (Figure 58–9A). It is thought that sensory neurons in the MOE detect volatile odors whereas those in the VNO detect nonvolatile chemosensory cues. Removal of the olfactory bulb, the only synaptic target of neurons in the MOE and the VNO, abolishes mating as well as aggression in mice and other rodents. These and other studies indicate an essential role for olfactory stimulation in initiating mating and fighting.

Genetically engineered disruption of pheromone responsiveness in the MOE or VNO reveals that these sensory tissues have a surprisingly complex role in the mating behavior of mice. A functional MOE is essential to trigger male sexual behavior, and an intact VNO is required for sex discrimination and directing the male to mate with females.

Key to these experiments is the fact that olfactory neurons in the MOE and the VNO use different signal transduction cascades to convert olfactory input into electrical responses.

The cation channel Trpc2 appears essential for pheromone-evoked signaling in VNO neurons; it is not expressed in MOE neurons, which use a different signal transduction apparatus. Thus mice lacking the gene trpc2 have a nonfunctional VNO and an intact MOE. Mating behavior directed to animals of the opposite sex appears unaltered in trpc2 mutant males as well as females. However, both males and female mutants often exhibit male sexual behavior with members of either sex. For example, trpc2 mutant females mate with females in a manner seemingly indistinguishable from wild type males, except of course the females cannot ejaculate. These and other findings suggest that the VNO is used to discriminate among sexual partners. When the VNO is inactivated animals can no longer distinguish between males and females, and mutants therefore exhibit male sexual behavior toward members of both sexes. Similarly, adult wild type females treated with testosterone also exhibit male sexual behavior toward females (Figure 58–9C).

One implication of these studies is that female mice possess the neural circuitry for male sexual behavior (Figure 58–9B). Activation of this neural circuit is inhibited in wild type females by sensory input from the VNO and by the lack of testosterone. Removal of the VNO or administration of testosterone activates male sexual behavior in females. Male mating behavior has been observed in females of many species, indicating that the findings in mice are likely to be of general relevance. Thus neural pathways for male and female mating behavior appear to be present in both sexes. The female-typical behavior of male rats following hypothalamic lesions is another example. In such cases it is the differential regulation of these circuits that underlies the sexually dimorphic expression of male and female sexual behaviors.

Early Experience Modifies Later Maternal Behavior

The preoptic area of the hypothalamus and the BNST are also important for another set of sexually dimorphic behaviors in females. Nursing rodents are good mothers, building a nest for their litter, crouching over the pups to keep them warm, and returning the pups to the nest when they happen to crawl away. Surgical lesioning of either the preoptic region or the BNST abolishes these maternal behaviors.

Studies of these behaviors have shed light on variations among individual females and how these differences exert lifelong effects on behavior of the offspring. Female lab rats exhibit distinct, stable forms of maternal care: Some lick and groom (LG) their pups frequently (high-LG mothers), whereas others lick and groom less frequently (low-LG mothers). Female offspring of high-LG mothers display high-LG activity when they themselves become mothers compared to female offspring of low-LG mothers (Figure 58–10). Moreover, pups of high-LG mothers show less anxiety-like behaviors in stressful conditions than do the pups of low-LG mothers.

These results suggested that levels of licking and grooming behavior and stress responses are genetically determined. However, studies by Michael Meaney and his colleagues provide an alternative explanation. When female rat pups are transferred from their mother to a foster mother at birth, their maternal behavior and stress responses as adults resemble those of their foster mother rather than those of their biological mother. Thus experience in infancy can lead to lifelong behavioral patterns. Because these patterns impact maternal behavior, their influence can endure over many generations.

How does brief and early experience lead to such long-lasting changes? One mechanism involves a covalent modification of the genome. Stress responses are coordinated by glucocorticoids acting on glucocorticoid receptors in the hippocampus. Throughout life tactile stimulation, including grooming, leads to transcriptional activation of the glucocorticoid receptor gene, which ultimately leads to reduced release of hypothalamic hormones that trigger stress responses. Tactile stimulation during early life also regulates the glucocorticoid receptor gene in a second way. A key site in the glucocorticoid receptor gene is methylated by the enzyme DNA methyltransferase, leading to gene inactivation. Initially gene methylation occurs in all pups, but pups reared by high-LG mothers are selectively demethylated. Thus in animals reared by high-LG mothers the effects of adult experience are potentiated. This is an example of epigenetic modification by which genes can be turned on or off more or less permanently. These animals exhibit blunted behavioral responses to stressful stimuli later in life.

What are the biological links between early experience and behavioral variation? A peptide hormone, oxytocin, plays a major role. Classical work showed that oxytocin regulates provision of milk by the mother, which occurs via reflex ejection in response to suckling (milk let-down). Oxytocin is synthesized by neurons in the hypothalamus and released into the general circulation through their projections in the posterior pituitary. It elicits smooth muscle contraction in the mammary gland, resulting in milk ejection. Oxytocin release from the pituitary is controlled by suckling, which provides a sensory stimulus that is conveyed to the hypothalamus by spinal afferent nerves.

Oxytocin and a related polypeptide hormone vasopressin also play important roles in regulating maternal bonding and other social behaviors (see Chapter 3). In these cases experience appears to modulate behaviors by affecting both release of oxytocin and levels of the oxytocin receptor in specific brain areas. In both rats and voles individual differences in the care females provide their offspring correlate with variations in oxytocin receptor level in specific brain areas. Especially noteworthy is that oxytocin receptor levels in several regions are higher in female offspring reared by high-LG mothers than in female progeny of low LG-mothers. Thus sensory stimulation may affect activity of the oxytocin and vasopressin systems, which in turn regulate maternal and other social behaviors.

These experiments suggest powerful mechanisms by which early experience can influence later behavior. Are they applicable to humans? Two recent observations suggest they are. First, as discussed in Chapter 56, children raised for lengthy periods in orphanages with little individual care have long-lasting defects in a variety of social behaviors. Even years after placement in foster homes these children have, on average, lower levels of oxytocin and vasopressin in their serum than children raised with biological parents. Second, people who have suffered abuse as children often grow up to be poor parents. Postmortem studies have shown that adults who had been abused as children exhibited greater promoter methylation of their glucocorticoid receptor genes than adults in control populations. Although these studies are new and require replication, they provide tantalizing hints at the biological mechanisms that underlie the lifelong effects of early parental care.

Are structural differences between the brains of male and female mammals also present in humans and, if so, might they be functionally important? Early studies revealed that a few structures are markedly larger in men: Onuf's nucleus in the spinal cord, the homolog of the spinal nucleus of the bulbocavernosus in rodents (Figure 58–7); the BNST, implicated in rodent mating behavior; and the interstitial nucleus of the anterior hypothalamus 3 (INAH3), related to the rodent SDN-POA discussed earlier (Figure 58–11).

Advances in high-resolution magnetic resonance imaging (MRI) and genetic technology have uncovered more subtle structural and molecular dimorphisms in the central nervous system. For example, structures such as the fronto-orbital cortex and several gyri—including the precentral, superior frontal, and lingual gyri—occupy a significantly larger volume in adult women compared to a cohort of adult men (Figure 58–12). Moreover, the frontomedial cortex, amygdala, and angular gyrus volumes are larger in men compared to women. Thus there are likely to be many sexual dimorphisms in the human brain.

What remains unclear is how these dimorphisms arise and how they relate to behavior. They might arise early from the organizational effects of hormones, or later as a result of experience. Structural differences arising before or soon after birth could underlie behavioral differences, whereas structural differences that arise later in life might be results of dimorphic behaviors.

Answers to these questions are fairly clear in a few cases. For example, studies of the development of neural circuits responsible for penile erection and lactation in rodents translate readily to humans. In contrast, differences in cognitive function, sexual partner preference, and gender identity are poorly understood. Little progress has been made in relating sex differences in cognitive functions to structural differences in the brain, in part because the very existence of cognitive differences remains a matter of controversy; if they exist at all, they are small and represent differences in means between highly variable male and female populations. On the other hand several lines of evidence have connected clear differences in gender identity and sexual orientation to dimorphisms in the brain.

Early insight into this issue came from observation of people with single-gene mutations that dissociate anatomical sex from gonadal and chromosomal sex, such as CAIS, CAH, and 5α-reductase deficiency (see Table 58–1). For example, girls with CAH experience an excess of testosterone during fetal life; the disorder is generally diagnosed at birth and corrected. Nevertheless, the early exposure to androgens is correlated with subsequent changes in gender-related behaviors. On average, girls with CAH tend to exhibit toy preferences and play typical of boys of equivalent age. There is also a small but significant increase in the incidence of homosexual and bisexual orientation in females treated for CAH as children, and a significant proportion of these females also express the desire to live as men, consistent with a change in gender identity. These findings suggest that early organizational effects of steroids affect gender-specific behaviors independent of chromosomal and anatomical sex.

In 5α-reductase II deficiency and CAIS many of the afflicted males show feminized external genitalia and are mistakenly raised as females until puberty. Thereafter their histories diverge. In 5α-reductase II deficiency the symptoms arise from a defect in testosterone processing largely confined to the developing external genitalia. At puberty a large increase in circulating testosterone virilizes the body hair, musculature, and most dramatically, the external genitalia. At this stage many but not all patients choose to adopt a male gender. In CAIS, in contrast, defects arise from a body-wide defect in the androgen receptor. These patients commonly seek medical advice after they fail to menstruate at puberty. Concordant with their feminized external phenotype, most CAIS patients have a female gender identity and a sexual preference for men. They opt for surgical removal of the testes and hormonal supplementation appropriate for females.

What accounts for the different outcomes? Among many possibilities, one is that the dramatic change in behavior in 5α-reductase II patients at puberty results from the effects of testosterone acting on the brain. In CAIS patients these effects do not occur because androgen receptors are absent from the brain. Clearly, however, this explanation does not rule out social and cultural upbringing as important factors in determining gender identity and sexual orientation.

A second set of studies probing the biology of sexual orientation assessed responses to pheromones. Pheromone perception in humans is quite different from that of mice, and is likely a less important sense. Humans do not have a functional VNO, and most of the genes implicated in pheromone reception in mice, such as trpc2 and those encoding VNO receptors, are absent (or nonfunctional pseudogenes) in the human genome. To the extent that humans do sense pheromones they appear to use the main olfactory epithelium and bulb. Chemicals that appear to be human pheromones include androstadienone (AND), an odorous androgenic metabolite, and estratetraenol (EST), an odorous estrogenic metabolite. AND is present at 10-fold higher concentrations in male sweat compared to female sweat, whereas EST is present in the urine of pregnant women. Both compounds can produce sexual arousal, AND in heterosexual women and EST in heterosexual men, even at concentrations so low that there is no conscious olfactory perception.

Brain areas activated by AND and EST have been identified by positron emission tomography (PET) imaging. When AND is presented, certain hypothalamic nuclei are activated in heterosexual women but not heterosexual men, whereas when EST is presented, adjacent regions containing clusters of nuclei are activated in men but not women (Figure 58–13A). In homosexual men and women there is a reversal of hypothalamic activation: AND but not EST activates hypothalamic centers in homosexual men, and conversely EST but not AND activates those areas in lesbian women. Heterosexual and homosexual brains therefore appear to process olfactory sensory information in different ways.

Do sexually dimorphic structures in homosexual brains correlate with anatomical sex or sexual orientation? Imaging studies have provided support for the view that the brains of homosexual men resemble those of heterosexual woman, and that the brains of homosexual women resemble those of heterosexual men (Figure 58–13B). Moreover, the volume of the sexually dimorphic BNST is small in male-to-female transsexuals compared to men, whereas female-to-male transsexuals appear to have a larger BNST compared to women (Figure 58–14). In these transgender individuals it remains possible that the structural dimorphism is a consequence rather than a cause of gender identity or sexual orientation. But they do raise the possibility that homosexual men and women are exposed prenatally to hormonal or other stimuli similar to those experienced by the other sex.

If prenatal influences do lead to dissociation of sex from gender, are those influences genetic? Other than the rare syndromes described earlier, attempts to find genetic bases for sexual orientation or gender identity have not been productive. Claimed genetic contributions are small and claims of associations with specific genomic loci have not been replicated. Thus, while the current weight of evidence favors some contribution of early, even prenatal, factors in these processes, their cause and relative weight remain unknown.

In most mammals, including humans, the sex determination pathway directs the differentiation of the testes in males and ovaries in females. The SRY gene on the Y chromosome is a master regulator that overrides a default female pathway to initiate formation of male gonads. The gonads then produce hormones that organize further differentiation of the body.

Of particular importance for generation of differences in the brain are the steroid hormones, including testosterone, estrogens, and progesterone. Again, the female pathway is often the default mode of differentiation, and at least during early life it is the absence of steroid hormones that permits feminization of brain and behavior to proceed. In many cases the underlying cellular phenomena resemble those that occur widely during normal development, such as apoptosis, neurite extension, synapse formation, and branch retraction. In sexually dimorphic structures these universal processes are brought under the control of sex steroids, so they occur to different extents in males and females. The effects of sex steroids are not confined to these irreversible organizational events during development; they also act in adult life to shape the behavioral repertoire of both sexes.

The link between sex differences in neuronal populations and behavior has been well established for behavioral responses such as erectile function and song production. Recent studies also provide insight into how individual variations in early experience are translated into adult variations in maternal behaviors. By contrast, we are only beginning to delineate the neural pathways underlying more complicated sexually dimorphic behaviors.

We do not yet understand how the nervous system generates various gender-related behaviors such as sexual orientation and gender identity. Here the relative contributions of genetic determinants and social experience remain to be determined. With advances in imaging and genetics, however, we are now poised to start linking specific neural pathways with sexually dimorphic complex social behaviors. These studies have the potential to yield insight into fundamental aspects of human nature such as our sexuality and the basis of many of our social interactions.