by Donald Pfaff
Exactly why might PPE gene expression have such a clear effect on lordosis behavior? Years earlier, Peg McCarthy, in the laboratory, had put forth the theory that certain neuropeptides can reduce the disruptive effects of stress. We applied Peg’s idea to the study of female PPE gene knockout (PPEKO) mice compared with their WT and heterozygous controls. PPEKO mice displayed alterations in fear and anxiety paradigms (Ragnauth et al. 2001). To examine the stability of responses, three squads of the genotypes were tested across seasons over a 20-month period. In a fear-conditioning paradigm, the PPEKO mice significantly increased freezing to both fear and fear plus shock stimuli relative to the controls. In the open-field test, the PPEKO mice spent significantly less time and traversed significantly less distance in the center of an open field than the WT controls. Further, PPEKO mice spent significantly less time and tended to be less active on the light side of a dark–light chamber than the controls, indicating that deletion of the enkephalin gene resulted in exaggerated responses to fear or anxiety-provoking environments. All our PPEKO data strongly suggested that enkephalin gene products act to inhibit fear and anxiety, and that they would release female reproductive behavior from disruption by stress.
Patterns of Genomic Contributions: α Compared with β
With respect to their regulation of behavior, the contributions of ER-α and ER-β are never the same (reviewed in Pfaff et al. 2002). As mentioned earlier, αERKO females never can perform lordosis behavior. The βERKO females actually have slightly higher lordosis behavior levels than their WT controls. The actions of ER-α gene products and ER-β actually oppose each other in the case of male aggression, which is vastly reduced in αERKO mice compared with WT controls, whereas βERKO males have higher aggression, especially just after puberty. With respect to molecular end points, the two genes, α and β, can have similar effects, such as in the induction of PR mRNA or in the reduction of ER mRNA. But with respect to behavioral actions, α and β effects are sometimes opposite and are always different.
As mentioned in Chapter 3, George Beadle and Edward Tatum, working with Neurospora fungi decades ago, won the Nobel Prize for their data supporting a “one gene—one enzyme” concept. In our case, with respect to ER-α and ER-β and the other genes covered in Chapter 3 and in this chapter, we obviously have proved that patterns of genes regulate patterns of instinctive behaviors.
The SRY Gene
Of course, in recounting the data from several molecular pharmacological studies using transgenic animals and microinjections of engineered viral vectors into brain, I am not ignoring the contribution of the Y chromosome to the masculinization and defeminization of behavior. In 1991, Robin Lovell-Badge, working at the Mill Hill laboratories of the Francis Crick Institute in London, discovered the SRY gene as the testis-determining gene. The SRY gene works through a cascade of Sox genes, eventually to derive the cells that will produce testosterone (for review, see Schaafsma and Pfaff 2014). In laboratory rodents, testosterone, acting in the brain just before birth and during the 24 hours after birth, has major behavioral effects that will last a lifetime. We used microarrays (Gagnidze, Pfaff, and Mong 2010) to show a large number of sex differences in hypothalamic and POA gene expression, some of which could play into these major sex differences in behavior. As a consequence, male laboratory animals will not perform lordosis behavior.
Genetic Contributions to Human Behavior Mediated through Several Steps
The gene / behavior causal relations I have summarized are strong and direct. Equally strong is another series of results that show gene / behavior causal relations that are much less direct. They have to do with the gene for GnRH and human sexual behavior in men: libido.
In Chapter 5, I will discuss GnRH in more detail. During the late 1980s we discovered that these neurons are born in the olfactory epithelium and make a long migration during development, finally entering the brain near their final functional place in the POA and anterior hypothalamus. We ended up being surprised about the role of this migration in human sexuality, a role proven by a genetic mutation leading to Kallmann’s syndrome.
Kallmann’s syndrome, which is X linked, is caused by genetic damage at Xp22.3, the Kall gene discovered by Christine Petit, in Paris. The syndrome is due to a failure of migration of GnRH neurons (Schwanzel-Fukuda, Bick, and Pfaff 1989). In a Kallmann’s patient the GnRH neurons are born normally in the olfactory epithelium, but after migrating up the nose they are dammed up at the top of the olfactory apparatus in the neurovascular structure, and they never reach the brain. We found that the Kall gene, whose damage causes X-linked Kallmann’s syndrome, codes for a cell-surface protein present during the migration of GnRH neurons into the brain. Importantly, men with Kallmann’s syndrome lack sexual desire—that is, they lack libido.
Putting these and closely related facts into logical order leads to a clear example of the complex participation of an individual gene in a human social behavior—sexual approach and attraction—through its actions during development. The gene / behavior causal relationship requires no less than six steps, each of which is proven beyond doubt. That is, males suffering from Kallmann’s syndrome lack sexual desire—behavioral libido is reduced 1) because of low testosterone levels, which occurs in turn 2) because of reduced gonadotropins (luteinizing hormone and follicle-stimulating hormone), which in turn are low 3) because the pituitary did not receive GnRH from the hypothalamus, 4) because no GnRH neurons are found in the brain, 5) because of a failure of GnRH neuronal migration, and 6) because of the absence of the cell surface protein produced by the Kall gene at Xp22.3.
Thus, it is possible to demonstrate with certainty a genetic influence on a crucial human social behavior, but the genetic effect works through six steps laid end to end.
Principle: Gene / Behavior Causal Relations
By the use of transgenic animals bearing gene knockouts and by the use of neuroanatomically specific microinjections of siRNA into well-chosen areas of the forebrain, Sonoko Ogawa and I were able to determine specific genes whose expression in particular neurons are essential for the performance of each of three types of behavior that regulate reproduction. For example, expression of the ER-α gene in the lateral POA is essential for female courtship behaviors. Then, expression of that same gene in VMH neurons is essential for mating behavior. Finally, expression of the ER-α gene in medial preoptic neurons is required for maternal behavior.
Wrapping up a Proof
Putting together the results in Chapters 1 through 4, we achieved the first demonstrations of specific molecular changes in particular neurons that drive a chain of behaviors of extraordinary biologic importance—and social behaviors, at that. For the simplest behavior, female mating behavior, we worked out the circuit that produces that social behavior. Chapter 1 laid out the neuroanatomy of hormone (estrogen)-binding neurons. Estrogen-dependent signals from those neurons regulate the performance of the lordosis circuit (Chapter 2), which was the first circuit worked out for a vertebrate behavior—specifically, a social behavior. Because ERs are ligand-activated transcription factors, we discovered the transcriptional logic of several effects of estrogens on mRNA levels in specific sets of neurons in the hypothalamus (Chapter 3). In turn, we could prove causal relations of some of these genes to lordosis and related behaviors (Chapter 4).
Further Reading
Choleris, E., J.-A. Gustafsson, K. S. Korach, L. J. Muglia, D. W. Pfaff, and S. Ogawa. 2003. “An Estrogen-Dependent Four-Gene Micronet Regulating Social Recognition: A Study with Oxytocin and Estrogen Receptor-Alpha and -Beta Knockout Mice.” Proceedings of the National Academy of Sciences of the United States of America 100: 6192–6197.
Choleris, E., S. Ogawa, M. Kavaliers, J.-A. Gustafsson, K. S. Korach, L. J. Muglia, and D. W. Pfaff. 2006. “Involvement of Estrogen Receptor Alpha, Beta and Oxytocin in Social Discrimination: A Detailed Behavioral Analysis with Knockout Female Mice.” Genes, Brain, and Behavior 5: 528–539.
Gagnidze, K., D. W. Pfaff, and J. A. Mong. 2010
. “Gene Expression in Neuroendocrine Cells during the Critical Period for Sexual Differentiation of the Brain.” Progress in Brain Research 186: 97–111.
Gagnidze, K., Z. M. Weil, L. C. Faustino, S. M. Schaafsma, and D. W. Pfaff. 2013. “Early Histone Modifications in the Ventromedial Hypothalamus and Preoptic Area following Oestradiol Administration.” Journal of Neuroendocrinology 25: 939–955.
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: 4751–4757.
Hunter, R. G., K. Gagnidze, B. S. McEwen, and D. W. Pfaff. 2015. “Stress and the Dynamic Genome: Steroids, Epigenetics and the Transposome.” Proceedings of the National Academy of Sciences of the United States of America 112: 6828–6833.
Hunter, R. G., K. J. McCarthy, T. A. Milne, D. W. Pfaff, and B. S. McEwen. 2009. “Regulation of Hippocampal H3 Histone Methylation by Acute and Chronic Stress.” Proceedings of the National Academy of Sciences of the United States of America 109: 17657–17662.
Hunter, R. G., B. S. McEwen, and D. W. Pfaff. 2013. “Environmental Stress and Transposon Transcription in the Mammalian Brain.” Mobile Genetic Elements 3 (2): e24555.
Hunter, R. G., G. Murakami, S. Dewell, M. Seligsohn, M. E. Baker, N. A. Datson, B. S. McEwen, and D. W. Pfaff. 2012. “Acute Stress and Hippocampal Histone H3 Lysine 9 Trimethylation, a Retrotransposon Silencing Response.” Proceedings of the National Academy of Sciences of the United States of America 109 (43): 17657–17662.
Kaplitt, M. G., A. D. Kwong, S. P. Kleopoulos, C. V. Mobbs, S. D. Rabkin, and D. W. Pfaff. 1994. “Preproenkephalin Promoter Yields Region-Specific and Long-Term Expression in Adult Brain following Direct in Vivo Gene Transfer via a Defective Herpes Simplex Viral Vector.” Proceedings of the National Academy of Sciences of the United States of America 91: 8979–8983.
Kaplitt, M. G., P. Leone, R. J. Samulski, X. Xiao, D. W. Pfaff, K. L. O’Malley, and M. J. During. 1994. “Long-Term Gene Expression and Phenotypic Correction Using Adeno-associated Virus Vectors in the Mammalian Brain.” Nature Genetics 8: 148–154.
Kaplitt, M. G., J. Pfaus, S. Kleopoulos, B. Hanlon, S. Rabkin, and D. W. Pfaff. 1991. “Expression of a Functional Foreign Gene in Adult Mammalian Brain following in Vivo Transfer via a Herpes Simplex Virus Type 1 Defective Viral Vector.” Molecular and Cellular Neurosciences 2: 320–331.
Magariños, A. M., and D. W. Pfaff. 2016. “Sexual Motivation in the Female and Its Opposition by Stress.” In Behavioral Neuroscience of Motivation. Vol. 27 of Current Topics in Behavioral Neuroscience. Edited by E. H. Simpson and P. D. Balsam. Berlin: Springer, 35–49.
Matsuda, K. I., H. Mori, B. M. Nugent, D. W. Pfaff, M. M. McCarthy, and M. Kawata. 2011. “Histone Deacetylation during Brain Development Is Essential for Permanent Masculinization of Sexual Behavior.” Endocrinology 152: 2760–2767.
McCarthy, M. M., S. P. Kleopoulos, C. V. Mobbs, and D. W. Pfaff. 1994. “Infusion of Antisense Oligodeoxynucleotides to the Oxytocin Receptor in the Ventromedial Hypothalamus Reduces Estrogen-Induced Sexual Receptivity and Oxytocin Receptor Binding in the Female Rat.” Neuroendocrinology 59: 432–440.
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.
Musatov, S., W. Chen, D. W. Pfaff, C. V. Mobbs, X. J. Yang, D. J. Clegg, M. G. Kaplitt, and S. Ogawa. 2007. “Silencing of Estrogen Receptor Alpha in the Ventromedial Nucleus of Hypothalamus Leads to Metabolic Syndrome.” Proceedings of the National Academy of Sciences of the United States of America 104: 2501–2506.
Nicot, A., S. Ogawa, Y. Berman, K. D. Carr, and D. W. Pfaff. 1997. “Effects of an Intrahypothalamic Injection of Antisense Oligonucleotides for Preproenkephalin mRNA in Female Rats: Evidence for Opioid Involvement in Lordosis Reflex.” Brain Research 777: 60–68.
Nomura, M., S. Andersson, K. S. Korach, J. A. Gustafsson, D. W. Pfaff, and S. Ogawa. 2006. “Estrogen Receptor-Beta Gene Disruption Potentiates Estrogen-Inducible Aggression but Not Sexual Behavior in Male Mice.” European Journal of Neuroscience 23: 1860–1868.
Nomura, M., L. Durbak, J. Chan, O. Smithies, J. A. Gustafsson, K. S. Korach, D. W. Pfaff, and S. Ogawa. 2002. “Genotype / Age Interactions on Aggressive Behavior in Gonadally Intact Estrogen Receptor Beta Knockout (βERKO) Male Mice.” Hormones and Behavior 41: 288–296.
Nomura, M., K. S. Korach, D. W. Pfaff, and S. Ogawa. 2003. “Estrogen Receptor Beta (ERbeta) Protein Levels in Neurons Depend on Estrogen Receptor Alpha (ERalpha) Gene Expression and on Its Ligand in a Brain Region-Specific Manner.” Molecular Brain Research 110: 7–14.
Ogawa, S., J. Chan, A. E. Chester, J.-A. Gustafsson, K. S. Korach, and D. W. Pfaff. 1999. “Survival of Reproductive Behaviors in Estrogen Receptor β Gene-Deficient (βERKO) Male and Female Mice.” Proceedings of the National Academy of Sciences of the United States of America 96 (22): 12887–12892.
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., A. E. Chester, S. C. Hewitt, V. R. Walker, J.-A. Gustafsson, O. Smithies, K. S. Korach, and D. W. Pfaff. 2000. “Abolition of Male Sexual Behaviors in Mice Lacking Estrogen Receptors α and β (αβERKO).” Proceedings of the National Academy of Sciences of the United States of America 97: 14737–14741.
Ogawa, S., V. Eng, J. Taylor, D. B. Lubahn, K. S. 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., D. B. Lubahn, K. S. Korach, and D. W. Pfaff. 1996. “Effects of Estrogen Receptor Gene Disruption on Aggressive Behaviors in Male Mice” [abstract]. Behavior Genetics 26 (6): 593.
______. 1997. “Behavioral Effects of Estrogen Receptor Gene Disruption in Male Mice.” Proceedings of the National Academy of Sciences of the United States of America 94: 1476–1481.
Ogawa, S., U. E. Olazábal, 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.
Ogawa, S., T. Washburn, J. Taylor, D. Lubahn, K. Korach, and D. W. Pfaff. 1998. “Modifications of Testosterone-Dependent Behaviors by Estrogen Receptor-Alpha Gene Disruption in Male Mice.” Endocrinology 139: 5058–5069.
Pfaff, D. W. 2001. “Precision in Mouse Behavior Genetics.” Proceedings of the National Academy of Sciences of the United States of America 98: 5957–5960.
Pfaff, D. W., S. Ogawa, H. K. Kia, N. Vasudevan, C. Krebs, J. Frohlich, and L.-M. Kow. 2002. “Genetic Mechanisms in Neural and Hormonal Controls over Female Reproductive Behavior.” In Hormones, Brain and Behavior, vol. 3. Edited by D. W. Pfaff, A. P. Arnold, A. M. Etgen, S. E. Fahrbach, and R. T. Rubin. San Diego: Academic Press / Elsevier, 3: 441–460.
Ragnauth, A. K., N. Devidze, V. Moy, K. Finley, A. Goodwillie, L.-M. Kow, L. J. Muglia, and D. W. Pfaff. 2005. “Female Oxytocin Gene-Knockout Mice, in a Semi-Natural Environment, Display Exaggerated Aggressive Behavior.” Genes, Brain, and Behavior 4: 229–239.
Ragnauth, A. K., A. Schuller, M. Morgan, J. Chan, S. Ogawa, J. Pintar, R. J. Bodnar, and D. W. Pfaff. 2001. “Female Preproenkephalin-Knockout Mice Display Altered Emotional Responses.” Proceedings of the National Academy of Sciences of the United States of America 98: 1958–1963.
Ribeiro, A. C., S. Musatov, L. 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: 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.
Romano, G. J., C. V. Mobbs, A. Lauber, R. D. Howells, and D. W. Pfaff. 1990. “Differential Regulation of Proenkephalin Gene Expression by Estrogen in the Ventromedial Hypothalamus of Male and Female Rats: Implications for the Molecular Basis of a Sexually Differentiated Behavior.” Brain Research 536: 63–68.
Schaafsma, S. M., and D. W. Pfaff. 2014. “Etiologies Underlying Sex Differences in Autism Spectrum Disorders.” Frontiers in Neuroendocrinology 35: 255–271.
Schwanzel-Fukuda, M., D. Bick, and D. W. Pfaff. 1989. “Luteinizing Hormone-Releasing Hormone (LHRH)–expressing Cells Do Not Migrate Normally in an Inherited Hypogonadal (Kallmann) Syndrome.” Molecular Brain Research 6: 311–326.