How the Vertebrate Brain Regulates Behavior

by Donald Pfaff

  Maternal behavior of female mice also depends on the normal expression of the ER-α gene. Female ER knockout mice showed markedly reduced maternal behaviors (Ogawa et al. 1998). This was primarily due to altered gene expression in the preoptic area, as microinjection of an adeno-associated viral vector bearing siRNA among preoptic area neurons mimicked the behavior-disrupting effect of a total ER-α gene loss (Ribeiro et al. 2012).

  In females, other behaviors as well depend on the gene for ER-α: the estrogenic effect to increase running wheel activity (likely connected with female-specific courtship behaviors) (Ogawa et al. 2003) and the estrogenic effect on feeding and weight gain (Geary et al. 2001). In general, the sex-specific effects of estrogens working through the gene product for ER-α can be conceived as maintaining a long chain of behaviors essential for successful reproduction: nutrition, courtship, copulation, and maternal behaviors.

  Regarding expression of two genes covered in Chapter 3, the progesterone receptor (PR) and preproenkephalin (PPE), studied at several levels of investigation, the sex differences are prominent (Pfaff, 1999) (Figure 9.1), and they contribute to the sex difference in lordosis behavior.

  Altogether, the functional results of these mechanisms produce females that are exquisitely sensitive to the hormonal sequence of estrogen priming followed by progesterone. This sequence drives lordosis behavior in the same time frame as the release of luteinizing hormone from the pituitary gland—thus, optimal timing. Equally important, the male lacks both of these functional results.

  An Alternative Mechanistic Route

  It should be noted that Arthur Arnold has found circumstances among birds and mammals in which testosterone levels during specific critical periods are not sufficient to explain all the sex differences in behavior. Based on his results and thinking, the discussion here of a purely genetic approach offers his alternative set of mechanisms. After that, applying them to clinically important sex differences will show their potential importance for human well-being.

  In the first place, Arnold points out that genes on the Y chromosome other than the testis-determining SRY gene can be expressed in neurons. Second, the fact that females have two X chromosomes and males only one leads to certain complications. Of course, the male has only the maternal X, whereas the female may have either the maternal or the paternal X to be inactivated in the service of “dosage compensation.” X-inactivation is a complex process. Some genes may actually escape X-inactivation and because many cells will have dosage-sensitive response systems, the inactivation escape can be crucial. In other cases, different neurons in the brain may have a balanced representation of genes from the maternal or the paternal X; or the representation could be unbalanced, with one or the other vastly predominating.

  Figure 9.1. Left: At several mechanistic levels, we see how transcription and function of the progesterone receptor (PR) work to produce lordosis behavior in the estrogen-primed female rat but not the male. (For references, see Pfaff, 1999, p. 124.) Right: Transcription and function of the neuropeptide enkephalin work to produce lordosis behavior in females but not males. (Adapted from Pfaff 1999; for references, see p. 129.)

  Third, Arthur Arnold reminds us of the pioneering work of Barry Keverne at the University of Cambridge who was the first to show parent-of-origin allelic imprinting of specific genes in the brain. In these cases, DNA methylation of a gene on the maternal X would prevent that gene from being expressed in at least some nerve cells in the male’s brain, whereas in the female the gene could be expressed from the paternal X. DNA methylation of the paternal X yields a simpler situation because the male does not even have it, whereas for a specific gene in the female brain, some neurons may be affected and others not.

  Fourth, we ask the question of what the inactivated X chromosome is actually doing in neurons of the female brain. If it is attracting epigenetic factors such as histone-modifying enzymes, then the concentrations of those factors on all the other chromosomes may be altered, with considerable consequences for behavior.

  It is important to think about this alternative set of mechanisms in the context of sexually differentiated brain disorders because the genetic thinking may suggest novel therapeutic approaches.

  Biological Import of Sex Differences

  Biologists legitimately ask the question, “Why do we have two sexes, anyway?” There are many theories out there, and it is beyond the scope of this book to examine them in extenso. However, there seem to be three main lines of thought, which are not mutually exclusive.

  First, some reasoning focuses on evolution. Regarding natural selection of the fittest, mixing strings of the male’s DNA with the female’s offers a much greater set of potential combinations than if such mixing did not occur. There are two results, both beneficial. 1) The offspring have a greater range of characteristics with which to meet the present environment. 2) As the environment changes, members of the species can change accordingly to better meet the challenges of the new environment.

  Second, most genetic mutations are deleterious. If there is only one copy of that gene, the individual is more susceptible to disease. Having more than one copy protects, especially since a recessive mutation will not be expressed after sexual reproduction during which the healthy gene copy from the sexual mate is dominant.

  Third, sexual reproduction requires that potential mates seek each other out. Male-male competition, combined with active mate choice by females, helps to guarantee reproduction that produces babies who will compete well in the next generation.

  Maladies: Neurodevelopmental Disorders and Autoimmune Disorders

  It has been popular in recent years to discuss the possibility of sex differences in a wide variety of animal and human behaviors. To avoid the possibility of error and to remain close to the physiological and molecular mechanisms, in my laboratory I have avoided wandering into areas of brain and behavior research for which the claimed sex difference is much less impressive than the overlap between sexes.

  Autism

  Many apparent sex differences in behavior are narrowly statistical, with large overlaps between male and female. One exception is autism, which is about four times more frequently found in males than females. Further, in addition to the psychological pain felt by many families who have an autistic child, the lifetime cost to society of that child will be as great as $4 million. These factors led me to seek collaborations with the developmental psychologist Sylvie Goldman and the pediatric neurologist Isabelle Rapin, who are experts in autism.

  A careful review of the literature led to our “three-hit” theory of the sex difference in autism (Pfaff, Rapin, and Goldman 2011). Briefly, 1) one hit is the sex difference of being a male; 2) the second hit is the large body of evidence that various forms of early stress predispose the child to autism; and 3) the third hit is the classic observation that monozygotic twins have much higher concordance rates for autism than dizygotic twins or just siblings, which points to genetic causes. I will cover all three topics briefly.

  Sexual differentiation. When I first began thinking about the sex difference in autism, I concentrated on the effects of testosterone on the brain, but Rockefeller postdoctoral researcher Sara Schafsma (2014), who was influenced by the pioneering thinking of Arthur Arnold, brought up other potential causal routes that were not mutually exclusive. As previously mentioned, Arnold had pointed to the incontrovertible fact that an additional genetic route to sex difference was derived simply from the fact that the female has two X chromosomes and the male only one. The resulting process that occurs only in females of X-inactivation and so-called dosage compensation is complex and allows for considerable variability.

  Further, we must consider the phenomenon of parent-of-origin allelic imprinting of specific genes, through which only the paternal copy or only the maternal copy of a specific gene is preferentially expressed in some nerve cells but not others. Finally, there is the possibility of a more subtle phenomenon called the “heterochromatin sink.” This idea poses that
the inactivated X-chromosome binds up epigenetic factors whose concentrations in the cell nucleus, as a result, are reduced, thus affecting gene expression from other chromosomes.

  All four of these sex differentiation routes could contribute to the large sex difference in autism.

  Early stress. Growing evidence suggests that exposure to prenatal adversity leads to neurological changes that underlie lifetime risks for mental illness (reviewed in Davis and Pfaff 2014). Beginning early in gestation, males and females show differential developmental trajectories and responses to stress. It is likely that the sex-dependent organization of neural circuits during the fetal period influences the differential vulnerability to mental health problems.

  Genetic mutations. At the beginning we all hoped that some small number of gene mutations would account for the heritability of autism. The postsynaptic anchoring protein Shank 3 was thought to be one strong candidate, as was the gene for the nerve cell membrane CTNAP2. But it turned out that mutations in these genes accounted only for an extremely small percentage of cases of autism. Recently, as reviewed by Washington University’s Evan Eichler, when counting de novo mutations it must be concluded that hundreds of genes’ mutations can contribute to the etiology of autism.

  Nevertheless, we wanted to test our three-hit theory of the sex difference in autism, and got the mouse bearing the CTNAP2 mutation from Daniel Geschwind’s laboratory at the University of California–Los Angeles. In one series of experiments, Sara Schaafsma and I tested the influence of each of the three hits on several measures of social and communicative behavior (Schaafsma et al. 2016). Hit one was male versus female. Hit two was prenatal stress versus control. Hit three was the CTNAP2 mutation versus wild type. Indeed, several of our behavioral results indicated that animals bearing all three hits (male, stressed, and mutation) had poorer communicative or social behavior than animals with no hits (female, nonstressed, and wild type).

  By saving brain tissue from every animal with behavioral measurements, we were able to link poorer behavioral results to deficiencies of corticotropin-releasing hormone receptor 1 (CRHR1) expression in the left hippocampus, and we found that the mRNA result in turn linked to epigenetic changes, acetylation on histone H3 and trimethylation on histone Hlysine4. We consider these results as constituting just one strand of evidence contributing to a much larger picture of molecular neurobiological changes contributing to autism in males.

  Autoimmune Disorders

  Females are more susceptible to autoimmune disorders, sometimes in the ratio of eight or nine to one. In such disorders, for example, either antibodies or T cells can attack self antigens, causing loss of function. There can be implications for behavior as well. Chronic fatigue syndrome, Sjögren’s syndrome, and fibromyalgia, for example, all affect a female’s behavior not only through pain but also through a loss of behavioral energy.

  An especially devastating condition is multiple sclerosis (MS). MS is a demyelinating disease that can be diagnosed via behavioral changes with or without brain imaging of lesions. Clinically, the loss of muscular control is obvious, but the disease is not necessarily progressive in a linear manner. Long periods of relapse may occur.

  The immunology involved is complicated, and heightened expression of certain immune phenotypes in females is thought to be involved, though this is not proven. Mouse models of experimental allergic encephalitis are being used to try to discover the most important underlying causes. Moreover, some data support Arthur Arnold’s approach, saying that simply having an XX sex chromosome complement (independent of ovarian versus testicular development) is part of the problem—that is, it is disease promoting.

  Principle inferred: The sex difference is strong for sex behaviors, and mechanisms of the difference are partly understood. For many more complex behaviors, sex differences may be claimed, but I have avoided studying them. An exception is autism, for which the sex difference is large and likely of medical importance.

  Further Reading

  Davis, E. P., and D. W. Pfaff. 2014. “Sexually Dimorphic Responses to Early Adversity: Implications for Affective Problems and Autism Spectrum Disorder.” Psychoneuroendocrinology 49: 11–25.

  Fink, G., D. W. Pfaff, and J. Levine. 2012. Handbook of Neuroendocrinology. San Diego: Academic Press / Elsevier.

  Gagnidze, K., D. W. Pfaff, and J. Mong. 2010. “Gene Expression in Neuroendocrine Cells during the Critical Period for Sexual Differentiation of the Brain.” Progress in Brain Research 186: 97–111.

  Geary, N., L. Asarian, K. S. Korach, D. W. Pfaff, and S. Ogawa. 2001. “Deficits in E2-Dependent Control of Feeding, Weight Gain and Cholecystokinin Satiation in ER-α Null Mice.” Endocrinology 142 (11): 4751–4757.

  Lauber, A. H., G. J. Romano, and D. W. Pfaff. 1991. “Sex Difference in Estradiol Regulation of Progestin Receptor mRNA in Rat Mediobasal Hypothalamus as Demonstrated by in Situ Hybridization.” Neuroendocrinology 53: 608–613.

  Musatov, S., W. Chen, D. W. Pfaff, M. G. Kaplitt, and S. Ogawa. 2006. “RNAi-Mediated Silencing of Estrogen Receptor α in the Ventromedial Nucleus of Hypothalamus Abolishes Female Sexual Behaviors.” Proceedings of the National Academy of Sciences of the United States of America 103: 10456–10460.

  Ogawa, S., J. Chan, J.-A. Gustafsson, K. S. Korach, and D. W. Pfaff. 2003. “Estrogen Increases Locomotor Activity in Mice through Estrogen Receptor Alpha: Specificity for the Type of Activity.” Endocrinology 144: 230–239.

  Ogawa, S., V. Eng, J. Taylor, D. Lubahn, K. Korach, and D. W. Pfaff. 1998. “Roles of Estrogen Receptor-Alpha Gene Expression in Reproduction-Related Behaviors in Female Mice.” Endocrinology 139: 5070–5081.

  Ogawa, S., U. E. Olazabal, I. S. Parhar, and D. W. Pfaff. 1994. “Effects of Intrahypothalamic Administration of Antisense DNA for Progesterone Receptor mRNA on Reproductive Behavior and Progesterone Receptor Immunoreactivity in Female Rat.” Journal of Neuroscience 14: 1766–1774.

  Ogawa, S., J. Taylor, D. B. Lubahn, K. S. Korach, and D. W. Pfaff. 1996. “Reversal of Sex Roles in Genetic Female Mice by Disruption of Estrogen Receptor Gene.” Neuroendocrinology 64: 467–470.

  Pfaff, D. W. 1999. Drive: Neurobiological and Molecular Mechanisms of Sexual Motivation. Cambridge, MA: MIT Press.

  Pfaff, D. W., I. Rapin, and S. Goldman. 2011. “Male Predominance in Autism: Neuroendocrine Influences on Arousal and Social Anxiety.” Autism Research 4 (3): 163–176.

  Pfaff, D. W., and R. E. Zigmond. 1971. “Neonatal Androgen Effects on Sexual and Nonsexual Behavior of Adult Rats Tested under Various Hormone Regimes.” Neuroendocrinology 7: 129–145.

  Ribeiro A. C., S. Musatov, A. Shteyler, S. Simanduyev, I. Arrieta-Cruz, S. Ogawa, and D. W. Pfaff. 2012. “siRNA Silencing of Estrogen Receptor-α Expression Specifically in Medial Preoptic Area Neurons Abolishes Maternal Care in Female Mice.” Proceedings of the National Academy of Sciences of the United States of America 109 (40): 16324–16329.

  Romano, G. J., A. Krust, and D. W. Pfaff. 1989. “Expression and Estrogen Regulation of Progesterone Receptor mRNA in Neurons of the Mediobasal Hypothalamus: An in Situ Hybridization Study.” Molecular Endocrinology 3: 1295–1300.

  Schaafsma, S., and D. W. Pfaff. 2014. “Etiologies Underlying Sex Differences in Autism Spectrum Disorders.” Frontiers in Neuroendocrinology 35: 255–271.

  Schaafsma, S., A. Reyes, K. Gagnidze, and D. W. Pfaff. 2016. “Testing a 3-Hit Theory of the Sex Difference in Autism: Experiments with CNTNAP2 Mutant Mice.” Proceedings of the National Academy of Sciences of the United States of America (in press).

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  SUMMARY

  This book summarizes how one field of science, neurobiology, has come to emerge as a scientifically important field. Within neuroscience, neuroendocrinology has proven to be the most effective approach to linking neurobiology to modern molecular biology.

  Putting some of our work together, we see how exploiting the discovery of hormone receptors in the brain contributed to working out the first circuit for a biologically crucial vertebrate b
ehavior and into the discovery of hormone effects on gene expression in the brain. In turn, analyzing an instinctive reproductive behavior turned out to be the best approach for revealing clear gene / behavior relationships. Fifty years of data have proven the reliability of the initial discovery and its subsequent developments.

  Throughout, rather than asking how the brain works, the book demonstrates a more successful approach: explaining how a biologically important function is physically realized. And the particular behavior that we have explained constitutes a mammalian social behavior.

  Brief Summary

  The book bridges explanations of how individual genes in single neuronal types participate in a newly understood neural circuit, to the production of an entire mammalian behavior. By linking genetics to neuroanatomy to physiology to behavior in a series of causal bridges, this book has offered a complete solution to the problem of how a vertebrate behavior can be produced and regulated.

  Our work began by discovering exact cellular targets for steroid hormones in the brain 50 years ago, with an emphasis on female-typical sex steroids. The limbic / hypothalamic system discovered is universal among vertebrate brains.