Geert J. De Vries

A Molecular Mechanism Regulating Rhythmic Output from the Suprachiasmatic Circadian Clock

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

A Model System for Study of Sex Chromosome Effects on Sexually Dimorphic Neural and Behavioral Traits

We tested the hypothesis that genes encoded on the sex chromosomes play a direct role in sexual differentiation of brain and behavior. We used mice in which the testis-determining gene (Sry) was moved from the Y chromosome to an autosome (by deletion ofSry from the Y and subsequent insertion of anSry transgene onto an autosome), so that the determination of testis development occurred independently of the complement of X or Y chromosomes. We compared XX and XY mice with ovaries (females) and XX and XY mice with testes (males). These comparisons allowed us to assess the effect of sex chromosome complement (XX vs XY) independent of gonadal status (testes vs ovaries) on sexually dimorphic neural and behavioral phenotypes. The phenotypes included measures of male copulatory behavior, social exploration behavior, and sexually dimorphic neuroanatomical structures in the septum, hypothalamus, and lumbar spinal cord. Most of the sexually dimorphic phenotypes correlated with the presence of ovaries or testes and therefore reflect the hormonal output of the gonads. We found, however, that both male and female mice with XY sex chromosomes were more masculine than XX mice in the density of vasopressin-immunoreactive fibers in the lateral septum. Moreover, two male groups differing only in the form of their Sry gene showed differences in behavior. The results show that sex chromosome genes contribute directly to the development of a sex difference in the brain.

The Epigenetics of Sex Differences in the Brain

Epigenetic changes in the nervous system are emerging as a critical component of enduring effects induced by early life experience, hormonal exposure, trauma and injury, or learning and memory. Sex differences in the brain are largely determined by steroid hormone exposure during a perinatal sensitive period that alters subsequent hormonal and nonhormonal responses throughout the lifespan. Steroid receptors are members of a nuclear receptor transcription factor superfamily and recruit multiple proteins that possess enzymatic activity relevant to epigenetic changes such as acetylation and methylation. Thus steroid hormones are uniquely poised to exert epigenetic effects on the developing nervous system to dictate adult sex differences in brain and behavior. Sex differences in the methylation pattern in the promoter of estrogen and progesterone receptor genes are evident in newborns and persist in adults but with a different pattern. Changes in response to injury and in methyl-binding proteins and steroid receptor coregulatory proteins are also reported. Many steroid-induced epigenetic changes are opportunistic and restricted to a single lifespan, but new evidence suggests endocrine-disrupting compounds can exert multigenerational effects. Similarly, maternal diet also induces transgenerational effects, but the impact is sex specific. The study of epigenetics of sex differences is in its earliest stages, with needed advances in understanding of the hormonal regulation of enzymes controlling acetylation and methylation, coregulatory proteins, transient versus stable DNA methylation patterns, and sex differences across the epigenome to fully understand sex differences in brain and behavior.

Ontogeny of the vasopressinergic neurons of the suprachiasmatic nucleus and their extrahypothalamic projections in the rat brain--presence of a sex difference in the lateral septum.

Immunocytochemical studies have revealed the presence of extensive vasopressinergic projections from the suprachiasmatic nucleus to the limbic system and other brain areas. Vibratome sections and the unlabeled antibody enzyme method were used to investigate the ontogeny of the vasopressinergic neurons of the suprachiasmatic nucleus and their exohypothalamic fibers in the rat brain. The first immunopositive neurons of this nucleus were revealed on the 2nd postnatal day. An adult appearance of the suprachiasmatic nucleus was detected on day 14. Although fibers appeared on the periventricular nucleus already on the 7th postnatal day, such fibers were visible in the lateral septum and lateral habenular nucleus only on day 10. From the 12th postnatal day onwards a marked sex difference developed with respect to the density of the vasopressin fibers in the lateral septum and, to a lesser extent, in the lateral habenular nucleus. In male rats the fiber density was higher in both areas. This sex difference persisted in adulthood.

Sex Differences in the Brain: The Not So Inconvenient Truth

### Introduction In 2001 the Institute of Medicine, a branch of the National Academy of Sciences in the U.S.A., concluded that many aspects of both normal and pathological brain functioning exhibit important yet poorly understood sex differences ([Wizemann and Pardu, 2001][1]). Ten years later, the

Sex differences in neurotransmitter systems.

Sex differences in behaviors and other centrally regulated processes have inspired research on structural differences in the brain that might underlie these functional differences. Structural sex differences have been found at almost every level in the brain (1-3). The manner in which such structural differences contribute to functional sex differences is only clear in some cases, notably in sex differences found in the spinal cord and lower brainstem, which contain motomeurons that innervate sexually dimorphic muscles. For example, rats have perineal muscles that are only present in males. These muscles contribute to erection, ejaculation and the deposition of a copulatory plug. They are innervated by motomeurons in the spinal dorsolateral nucleus and the nucleus of the bulbocavemosus, which contain about three times as many motomeurons in males as in females (4). It is more difficult to understand how structural sex differences at higher levels of the brain contribute to functional differences, even when such differences are found in areas implicated in sexually differentiated functions. For example, in songbirds, males typically sing, but females do not. This difference seems to be reflected in song control nuclei which, in males compared to females, are generally larger with more and bigger cells that have more extensive dendritic trees covered with more synapses (5). How most of the song nuclei contribute to song is, however, still largely unknown. Even less well understood are the well documented sex differences in the medial preoptic area (POA) of the rat in, for example, the size of nuclei, cell density, arborization and connectivity (6). Since the POA has been implicated in the regulation of male sexual behavior, it has been suggested that these differences might underlie sex differences in the regulation of male sociosexual behavior, but this suggestion has never been substantiated (6, 7). At least three factors make it difficult to relate structural sex differences in the brain to sexually dimorphic functions. First, sexually dimorphic structures are always found in areas implicated in more than one function. For example, the POA not only regulates male sociosexual behavior, but also female sexual behavior, gonadotropin release, body temperature, osmolarity of extracellular fluids, and sleep rhythms (8). Since many of these functions are sexually differentiated, it is hard to estimate the relative impact of the observed sex differences on each of these functions. Secondly, the connectivity of sexually dimorphic areas is often poorly documented. It is, therefore, often unknown which other brain areas might be influenced by a given sex difference. For example, the medial preoptic nucleus (MPN) of the rat may be one of the best studied sexually dimorphic areas in the brain, yet, many questions remain about its connections. Its central part, which is live times larger in males than in females, is still so small that it is almost impossible to limit tracer injections to that part (9, 10). Also, many questions remain as to the connections of the different neurotransmitter systems that either originate in, or innervate the MPN (11). Thirdly, not many of the reported structural sex differences are dramatically influenced by sex steroids in adulthood, although many of the sexually differentiated functions are so influenced (7). This makes it more difficult to identify which cellular systems in a sexually dimorphic area could contribute to functional sex differences. For example, the size of the central part of the MPN depends on the perinatal presence or absence of androgens, as does the tendency of a rat to display male sexual behavior. However, whereas the size of the central part is not notably influenced by castration in adulthood (6), male sexual behavior is strongly influenced. Studying the neurotransmitter systems that originate in, or innervate sexually dimorphic areas may help in relating structure to function, as many neurotransmitter systems are sensitive to sex steroid levels in adulthood. Neurochemical studies have revealed that, in certain areas, neurotransmitter synthesis, content and metabolism is sexually differentiated and under the influence of sex steroids in adulthood (12, 13). The results of such studies could, therefore, suggest which neurotransmitters might be involved in sexually dimorphic functions. However, since such studies often use homogenized brain tissue, they lack adequate anatomical resolution and, consequently, fail to close the gap between structure and function. This is rapidly changing, now neurotransmitter synthesis, content and receptors can be studied with histochemistry, immunocytochemistry, in situ hybridization and receptor autoradiography. These techniques have revealed sex steroid effects on neurotransmitter systems in development and adulthood that can be compared to similar effects of sex steroids on function regulated by the brain. Their anatomical

Neural Connections of the Anterior Hypothalamus and Agonistic Behavior in Golden Hamsters

In male golden hamsters, offensive aggression is regulated by an interaction between arginine-vasopressin and serotonin at the level of the anterior hypothalamus. The present studies were conducted to study a neural network underlying this interaction. The connections of the anterior hypothalamus were examined by retrograde and anterograde tracing in adult male hamsters. Several limbic areas were found to contain both types of tracing suggesting reciprocal connections with the anterior hypothalamus. Their functional significance relating to the consummation of aggression was tested by comparing neuronal activity (examined through quantification of c-Fos-immunolabeling) in two groups of animals. Experimental animals were sacrificed after attacking an intruder. Control animals were sacrificed after exposure to a woodblock carrying the odor of an intruder that elicited behaviors related to offensive aggression without its consummation. An increased density of Fos-immunoreactivity was found in experimental animals within the medial amygdaloid nucleus, ventrolateral hypothalamus, bed nucleus of the stria terminalis and dorsolateral part of the midbrain central gray. These data suggest that these areas are integrated in a neural network centered on the anterior hypothalamus and involved in the consummation of offensive aggression. Finally, c-Fos-immunoreactivity was combined with labeling of serotonin and vasopressin neurons to identify sub-populations particularly associated with offensive aggression. Vasopressin neurons in the nucleus circularis and medial division of the supraoptic nucleus showed increased neuronal activity in the fighters, supporting their role in the control of offensive aggression.

Masculine Sexual Behavior Is Disrupted in Male and Female Mice Lacking a Functional Estrogen Receptor α Gene

Masculine sexual behavior is regulated by testosterone (T). However, T can be metabolized to form estrogens or other androgens, which then activate their own receptors. We used knockout mice lacking a functional estrogen receptor alpha (ER alpha) gene to test the hypothesis that, following aromatization, T acts via the ER alpha to activate normal masculine sexual behavior. After gonadectomy and T replacement, wild-type (WT) male and female mice displayed masculine behavior. However, given the same T treatment, little masculine behavior was displayed by mice of either sex that lack a normal copy of the ER alpha gene. In particular, the latency to display masculine sex behavior and the number of mount attempts per trial were significantly reduced in the ER alpha- mice compared to WT littermates (P < 0.05). In addition, we found that in both sexes, ER alpha- mice have a smaller cluster of androgen receptor immunoreactivity in the bed nucleus of the stria terminalis. Using adult ER alpha- mice we were unable to determine whether these genotypic differences are due to organizational or activational effects. However, it is clear that the ER alpha plays a key role in the expression of masculine sexual behavior and in the regulation of androgen receptors in a neuronal cell population involved in the display of motivated behaviors.

Anatomy, Development, and Function of Sexually Dimorphic Neural Circuits in the Mammalian Brain

Publisher Summary This chapter discusses the anatomy, development, and function of sexually dimorphic neural circuits in the mammalian brain. If structural sex differences promote as well as prevent sex differences in behavior and other centrally regulated functions in rodents, this may be true for human brains as well. Human brains show sex differences in the volume of several brain structures, including the anterior commissure, the left planum temporale, and several nuclei in the hypothalamus and BST. In addition, several neurotransmitter systems are sexually dimorphic. Although these differences are typically associated with differences in behavior, these sex differences may also normalize behavior in males and females. The effects of stroke support the possibility that similar functions indeed have a sexually dimorphic neural basis. Stroke affects language abilities differently in males than it does in females. Frontal lesions cause aphasias more frequently in females than in males, whereas the opposite is true for temporal lesions.

Chapter 1.1 Anatomy and function of extrahypothalamic vasopressin systems in the brain

The most prominent sites of vasopressin (VP) production in the rat brain are the paraventricular nucleus, the supraoptic nucleus, the suprachiasmatic nucleus, the bed nucleus of the stria terminalis (BST), and the medial amygdaloid nucleus (MA). Recently a number of new sites have been suggested, including the hippocampus, the diagonal band of Broca, and the choroid plexus. This chapter shows how differential regulation of these VP systems can be exploited to identify the contributions of individual VP systems to the various central functions in which VP has been implicated. It will focus on the development, anatomy, and function of the sexually dimorphic VP projections of the BST and MA. This system contains more cells and has denser projections in males than in females. This system is also extremely responsive to gonadal steroids as it only produces VP in the presence of gonadal steroids. It has been implicated in sexually dimorphic functions such as aggressive behavior as well as in non-sexually dimorphic functions such as social recognition memory. Using comparative studies done in prairie voles as an example, this chapter makes the case that the VP projections of the BST and MA may simultaneously generate sex differences in some brain functions and behaviors and prevent them in others.