How the Vertebrate Brain Regulates Behavior

by Donald Pfaff

  Thinking of our competitive ER / TR binding theory, Rod Scott, a molecular endocrinologist trained in Glasgow, investigated the activity of an ERE identified in the PR proximal promoter and its interactions with the ER and TR (Scott et al. 1997). In addition, we compared ER and TR interactions on the PR ERE construct with that of the vitellogenin A2 consensus ERE. Electrophoretic mobility shift assays demonstrated that TR does indeed bind to the PR ERE as well as to the consensus ERE sequence in vitro. Further, these two EREs were differentially regulated by T3 in the presence of TR. T3 in the presence of TR-α increased transcription from a PR ERE construct approximately fivefold and had no inhibitory effect on estrogen induction. Similarly, T3 also activated a β-galactosidase reporter construct containing PR promoter sequences spanning −1400 to +700. In addition, the TR isoforms β1 and β2 also stimulated transcription from the PR ERE construct by fivefold to sixfold. A TR α mutant lacking the ability to bind AGGTCA sequences in vitro failed to activate transcription from the PR ERE construct, demonstrating dependence on DNA binding. In contrast to its actions on the PR ERE construct, TR-α did not activate transcription from the vitellogenin A2 consensus ERE but rather attenuated estrogen-mediated transcriptional activation. Surprisingly, attenuation from the vitellogenin A2 consensus ERE is not necessarily dependent on DNA binding, as the TR-α DNA binding mutant was still able to inhibit estrogen-dependent transactivation.

  As with Larissa Faustino’s results, our competitive DNA binding theory did not account for all of our data. Instead, divergent pathways must exist for activation and inhibition by TR. Because ER, PR, and TR are all present in VMH neurons, these findings must indicate neuronal integration (regarding TR, probably relevant to the seasonality of reproduction), important for the regulation of female reproductive behavior.

  When Mitsuhiro Kawata came to the laboratory, I was re-exposed to traditional Japanese loyalty. Mitsuhiro (“Mike”) visited the graves of famous Japanese scientists who had worked in the United States, continued to practice kendo (a Japanese sword-fighting discipline), and showed me how to make Japanese tea in the correct manner (using traditional Japanese pottery). After he returned to Kyoto he became professor of neuroanatomy and, as head of his department there, led quite an ambitious study of sexual development (Matsuda et al. 2011). He knew that epigenetic histone modifications are emerging as important mechanisms for conveyance of and maintenance of the effects of the hormonal milieu on the developing brain. Thus, he hypothesized that alteration of histone acetylation status early in development by sex steroid hormones would be important for sexual differentiation of the brain.

  His team in Kyoto found that during the critical period for sexual differentiation, histones associated with promoters of essential genes in masculinization of the brain (ER-α and aromatase enzymes) in the medial preoptic area, an area necessary for male sexual behavior, were differentially acetylated between the sexes. Consistent with these findings, binding of histone deacetylase (HDAC) 2 and 4 to the promoters was higher in males than in females. Most importantly, to examine the involvement of histone deacetylation on masculinization of the brain at the behavioral level, his team inhibited HDAC in vivo by intracerebroventricular infusion of the HDAC inhibitor trichostatin A or antisense oligodeoxynucleotide directed against the mRNA for HDAC2 and HDAC4 in newborn male rats. Aspects of male sexual behavior in adulthood were significantly reduced by administration of either trichostatin A or antisense oligodeoxynucleotide. These results demonstrated that histone modifications in the brain play a role in the sexual differentiation of behavior.

  Kawata’s result represented a specific example of what Cambridge University biologist Barry Keverne and I mean when we talk about epigenetic effects in the developing brain (reviewed in Keverne, Tabansky, and Pfaff 2015). Now, in 2016, we have convincing examples of DNA methylation effects and noncoding RNA effects as well as important histone N-terminus modifications.

  One of the consequences of neonatal androgen exposure during the neonatal critical period for sexual differentiation is the absence of lordosis in the male. Rod Scott, mentioned earlier, came to the laboratory and investigated sex differences with respect to the PR promoter (Scott, Wu-Peng, and Pfaff 2002). He measured protein binding on the PR ERE and mRNA levels for PR-A (the shorter transcript) and PR-B (the longer transcript) and compared the results between female and male rats. In both sexes, protein extracts demonstrated an increase in nuclear protein binding activity to a PR ERE after estradiol treatment. However, females’ ERE protein binding was greater than males’. In both cases, reflecting the binding data, estradiol pretreatment led to an increase in PR-B messenger RNA, although this increase was significantly larger in females than in males. Estradiol treatment also led to a significant increase in specific binding of hypothalamic nuclear proteins to the PR ERE from both female and male hypothalamic extracts. The predominance of PR-B over PR-A mRNA in the rat hypothalamus and pituitary, and the quantitative differences between female and male rats could both contribute to the greater responsiveness of female rats to progesterone with respect to control over luteinizing hormone release from the pituitary and lordosis behavior.

  Rod Scott further analyzed the PR promoter using a construct made from a herpes simplex viral vector (Scott et al. 2003). That is, to demonstrate that estradiol (E) induces transcription via the PR promoter, and to identify sequences within the PR promoter responsible for tissue-specific and hormonal regulation, he used a defective herpes simplex virus vector for direct gene transfer into the rat pituitary and brain. A viral amplicon expressing the β-galactosidase gene (for a reporter, lacZ) under the regulation of a 2.1-kilobase PR promoter fragment to create a defective viral vector for gene transfer was microinjected into the brain. In the pituitary, lacZ activity was observed in cells of the anterior lobe. However, no activity was seen in the neurointermediate lobe, demonstrating tissue-specific transcriptional regulation. An approximately six times increase in cells demonstrating β-galactosidase activity was observed in the anterior lobe after treatment with estrogen. Likewise, injection of defective viral vector into the hypothalamus followed by treatment with estrogen resulted in an approximately eight times increase in cells demonstrating β-galactosidase activity including the VMH, the very nerve cell group responsible for estrogen-dependent reproductive behavior.

  Finally, a more complex point about relations among hormone-signaling neuroendocrine systems: female sexual behaviors are inhibited by stress (reviewed in Magariños and Pfaff 2016). Ironically, it was the stress effect on histone modifications that led us into the cooperation with Dave Allis’s laboratory to begin with. Richard Hunter showed the effect of acute restraint stress on transcriptional-repressive histone methylation in hippocampal neurons (Hunter et al. 2009). He later conceived hippocampal histone H3 lysine 9 trimethylation as suppressing transcription of potentially disruptive transposons (Hunter, McEwen, and Pfaff 2013; Hunter et al. 2012, 2015)—these are the “jumping genes” of Nobel laureate Barbara McClintock.

  How Multiple Transcriptional Systems Overdetermine Reproductive Behavior and Synchronize It with Ovulation

  First of all, we have several interesting examples of potential multiplicative effects of estrogens on transcriptional systems in VMH neurons: 1) both GnRH and the GnRH receptor, 2) both OT and the OT receptor, 3) both tyrosine hydroxylase for the production of norepinephrine and the α1-receptor, and 4) both enkephalin and the δ-opioid receptor showed estrogen sensitivity. Obviously, when both the ligand and the receptor are increased after estrogen treatment, the two hormone effects could multiply. On top of that, the overall trophic effects of estrogens in VMH neurons, as documented earlier, have the potential to multiply all VMH outgoing signals.

  Further, we have the concept of separate but converging contributions of individual transcriptional systems to regulate lordosis behavior and thus all of the female’s contributions to reproduction, because lordosis is required for fertilization.

  Consider the eight transcriptional
systems I reviewed early in this chapter (Figure 3.1). 1) The PR induction amplifies the estrogen effects because of downstream genes regulated by PR. Additionally, PR induction brings into play the same hormone, progesterone, used to control the ovulatory surge of LH from the pituitary, thus helping to synchronize ovulation and lordosis. The female should not be exposed to predation, as required by mating, if that mating would not be productive of a litter. 2) Likewise, GnRH working through the GnRH receptor promotes mating behavior and is also required for the ovulatory surge of LH, thus synchronizing mating and ovulation. 3) Estrogens enhance the transcription of neuronal nNOS. Release of NO from these neurons stimulates the pulsatile release of GnRH, which not only induces LH release to induce ovulation but also fosters lordosis. Again, there is a synchrony between the endocrine and the behavioral requirements for reproduction.

  In addition, NO coordinates with glutamatergic neurons to heighten excitability and thus amplify the VMH effect. 4–5) Both adrenergic inputs and cholinergic inputs are neurotransmitters used by ascending brain arousing systems, serving an arousal state necessary for the initiation of mating behavior. Estrogen in the absence of arousal does not work to facilitate lordosis (see Chapter 8). 6) OT working through the OT receptor (Chapter 6) reduces the anxiety-provoking effects of environmental stress: anxiolysis. The female must leave the burrow in order to mate, thus exposing herself to predation. Anxiolysis reduces the behavior-disrupting effects of this stress. 7) Transcription for the neuropeptide enkephalin working through its δ-opioid receptor, both increased by estrogens, can be seen as important for the induction of analgesia. Not only must the female be able to mate whether or not in some degree in pain, but also small females accosted by large males must allow mating to go forward. 8) And all these effects are amplified by the trophic actions of estrogens, as previously detailed, on VMH neurons.

  Putting these findings in historical perspective, I note that decades ago George Beadle and Edward Tatum worked with Neurospora and won the Nobel Prize for their “one gene, one enzyme” concept. Now, working with mammalian reproductive behaviors, I can see clearly that we have patterns of genes governing the expression of patterns of behavior.

  From this overwhelming list of molecular links between estrogen administration, transcription of specific mRNAs in hypothalamic neurons, and lordosis behavior, it appears clearly that in transcriptional terms lordosis is “overdetermined” by estrogenic actions in VMH neurons. No chances of failure have been risked.

  Principle inferred: Several transcriptional systems participate to overdetermine the performance of female reproductive behavior at the time of ovulation. I have conceived how their separate contributions dovetail to contribute to the behavior’s regulation. One step deeper, we use epigenetic methodologies to investigate how those transcriptional mechanisms are themselves controlled. In the next chapter I will show how this field of work was the first to establish causal gene / behavior relations in the vertebrate brain.

  Further Reading

  Ceccatelli, S., L. Grandison, R. E. M. Scott, D. W. Pfaff, and L.-K. Kow. 1996. “Estradiol Regulation of Nitric Oxide Synthase mRNAs in Rat Hypothalamus.” Neuroendocrinology 64: 357–363.

  Chung, S. R., J. T. McCabe, and D. W. Pfaff. 1991. “Estrogen Influences on Oxytocin mRNA Expression in Preoptic and Anterior Hypothalamic Regions Studied by in Situ Hybridization.” Journal of Comparative Neurology 307: 281–295.

  Chung, S. R., D. W. Pfaff, and R. S. Cohen. 1988. “Estrogen-Inducted Alterations in Synaptic Morphology in the Midbrain Central Gray.” Experimental Brain Research 69: 522–530.

  ______. 1990a. “Projections of Ventromedial Hypothalamic Neurons to the Midbrain Central Gray: An Ultrastructural Study.” Neuroscience 38: 395–407.

  ______. 1990b. “Transneuronal Degeneration in the Midbrain Central Gray following Chemical Lesions in the Ventromedial Nucleus: A Qualitative and Quantitative Analysis.” Neuroscience 38 (2): 409–426.

  Cohen, R. S., S. R. Chung, and D. W. Pfaff. 1984. “Alteration by Estrogen of the Nucleoli in Nerve Cells of the Rat Hypothalamus.” Cell and Tissue Research 235: 485–489.

  Cohen, R. S., and D. W. Pfaff. 1981. “Ultrastructure of Neurons in the Ventromedial Nucleus of the Hypothalamus in Ovariectomized Rats with or without Estrogen Treatment.” Cell and Tissue Research 217: 451–470.

  Commons, K. G., and D. W. Pfaff. 2001. “Ultrastructural Evidence for Enkephalin Mediated Disinhibition in the Ventromedial Nucleus of the Hypothalamus.” Journal of Chemical Neuroanatomy 21: 53–62.

  Davis, P. G., B. McEwen, and D. W. Pfaff. 1979. “Localized Behavioral Effects of Tritiated Estradiol Implants in the Ventromedial Hypothalamus of Female Rats.” Endocrinology 104: 898–903.

  Dellovade, T. L., H. K. Kia, Y.-S. Zhu, and D. W. Pfaff. 1999a. “Thyroid Hormones and Estrogen Affect Oxytocin Gene Expression in Hypothalamic Neurons.” Journal of Neuroendocrinology 11: 1–10.

  ______. 1999b. “Thyroid Hormone Coadministration Inhibits the Estrogen-Stimulated Elevation of Preproenkephalin mRNA in Female Rat Hypothalamic Neurons.” Neuroendocrinology 70: 168–174.

  Dellovade, T. L., Y.-S. Zhu, L. Krey, and D. W. Pfaff. 1996. “Thyroid Hormone and Estrogen Interact to Regulate Behavior.” Proceedings of the National Academy of Sciences of the United States of America 93: 12581–12586.

  Devidze, N., J. A. Mong, A. M. Jasnow, L.-M. Kow, and D. W. Pfaff. 2005. “Sex and Estrogenic Effects on Co-expression of mRNAs in Single Ventromedial Hypothalamic Neurons.” Proceedings of the National Academy of Sciences of the United States of America 102 (40): 14446–14451.

  Faustino, L. C., K. Gagnidze, T. Ortiga-Carvalho, and D. W. Pfaff. 2015. “Impact of Thyroid Hormones on Estrogen Receptor-Alpha-Dependent Transcriptional Mechanisms in Ventromedial Hypothalamus and Preoptic Area.” Neuroendocrinology 101: 331–346.

  Gagnidze, K., and D. W. Pfaff. 2016. “Epigenetic Mechanisms: DNA Methylation and Histone Protein Modification.” In Neuroscience in the 21st Century: From Basic to Clinical. 2nd edition. Edited by D. W. Pfaff and N. D. Volkow. Berlin: Springer, 1939–1978.

  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.

  Gagnidze, K., Z. M. Weil, and D. W. Pfaff. 2010. “Histone Modifications Proposed to Regulate Sexual Differentiation of Brain and Behavior.” Bioessays 32 (11): 932–939.

  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 (22): 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.

  Hunter, R. G., D. W. Pfaff, and B. S. McEwen. 2016. “Epigenetic Effects of Stress on the Mitochondrial Genome.” Proceedings of the National Academy of Sciences of the United States of America (in press).

  Jones, K. J., D. M. Chikaraishi, C. A. Harrington, B. S. McEwen, and D. W. Pfaff. 1986. “In Situ Hybridization Detection of Estradiol-Induced
Changes in Ribosomal RNA Levels in Rat Brain.” Brain Research: Molecular Brain Research 1: 145–152.

  Jones, K. J., C. A. Harrington, D. M. Chikaraishi, and D. W. Pfaff. 1990. “Steroid Hormone Regulation of Ribosomal RNA in Rat Hypothalamus: Early Detection Using in Situ Hybridization and Precursor-Product Ribosomal DNA Probes.” Journal of Neuroscience 10 (5): 1513–1521.

  Jones, K. J., B. S. McEwen, and D. W. Pfaff. 1988. “Quantitative Assessment of Early and Discontinuous Estradiol-Induced Effects on Ventromedial Hypothalamic and Preoptic Area Proteins in Female Rat Brain.” Neuroendocrinology 48: 561–568.

  Jones, R. J., D. W. Pfaff, and B. S. McEwen. 1985. “Early Estrogen-Induced Nuclear Changes in Rat Hypothalamic Ventromedial Neurons: An Ultrastructural and Morphometric Analysis.” Journal of Comparative Neurology 239: 255–266.

  Kaufman, L. S., B. S. McEwen, and D. W. Pfaff. 1988. “Cholinergic Mechanisms of Lordotic Behavior in Rats.” Physiology and Behavior 43: 507–514.

  Keverne, E. B., D. W. Pfaff, and I. Tabansky. 2015. “Epigenetic Changes in the Developing Brain: Effects on Behavior.” Proceedings of the National Academy of Sciences of the United States of America 112: 6789–6795.

  Kow, L.-M., A. E. Johnson, S. Ogawa, and D. W. Pfaff. 1991. “Electrophysiological Actions of Oxytocin on Hypothalamic Neurons in Vitro: Neuropharmacological Characterization and Effects of Ovarian Steroids.” Neuroendocrinology 54: 526–535.

  Kow, L.-M., A. W. Lee, C. Klinge, J.-A. Gustafsson, and D. W. Pfaff. 2016. “Molecular and Cellular Mechanisms for Estrogenic Effects on Brain and Behavior.” In Hormones, Brain and Behavior. 3rd edition. Edited by D. W. Pfaff and M. Joels. Cambridge: Elsevier, in press.

  Kow, L.-M., and D. W. Pfaff. 1985. “Estrogen Effects on Neuronal Responsiveness to Electrical and Neurotransmitter Stimulation: An in Vitro Study on the Ventromedial Nucleus of the Hypothalamus.” Brain Research 347: 1–10.