by Donald Pfaff
Not only are telencephalic cell groups not necessary for lordosis, but they have a net inhibitory effect. The lordosis circuit as presented in Figure 2.6 produces the behavior, effectively releasing the behavior from cortical inhibition.
Hypothalamic Module
Obviously the main job of the hypothalamic module is to add hormone dependence to the behavior, by virtue of which the hypothalamic module is necessary for the rest of the circuit to work. At the same time, hypothalamic neurons coordinate female reproductive behavior with ovulation. The synchrony of lordosis with ovulation clearly is biologically adaptive, helping to ensure that mating events will be productive.
We got lucky with respect to the properties of the VMH cells in the hypothalamic module. The estrogen receptors I had discovered in brain (Chapter 1) turned out to be ligand-activated transcription factors (estrogen-dependent genes transcribed consequent to estrogens binding to ER-α). This fact allowed us to use the techniques of molecular biology such as in situ hybridization and polymerase chain reaction to study gene expression related to lordosis in VMH cells, followed by epigenetic explorations of histone N terminus modifications (Chapter 3), followed by determination of gene / behavior relationships (Chapter 4).
In summary, we started with the information I had discovered about how hormones impact the brain (Chapter 1). In Chapters 3 and 4 we will be able to link these hormones to gene expression and genomic influences on behavior. Here, in Chapter 2, I have detailed a manifestation of lordosis behavior mechanisms in physical terms.
Principle inferred: Yes, it is possible to work out the circuit for producing a complete, normal vertebrate behavior. The circuit for this social behavior, essential for reproduction, is hierarchical, composed of modules. Each module adds a unique regulatory feature.
Further Reading
Brink, E. E., D. T. Modianos, and D. W. Pfaff. 1980. “Ablations of Lumbar Epaxial Musculature: Effects on Lordosis Behavior of Female Rats.” Brain, Behavior and Evolution 17: 67–88.
Brink, E. E., J. I. Morrell, and D. W. Pfaff. 1979. “Localization of Lumbar Epaxial Motoneurons in the Rat.” Brain Research 170: 23–41.
Brink, E. E., and D. W. Pfaff. 1980. “Vertebral Muscles of the Back and Tail of the Albino Rat (Rattus norvegicus albinus).” Brain, Behavior and Evolution 17 (1): 1–47.
______. 1981. “Supraspinal and Segmental Input to Lumbar Epaxial Motoneurons in the Rat.” Brain Research 226: 43–60.
Chung, S. R., D. W. Pfaff, and R. S. Cohen. 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, M. S., S. Schwartz-Giblin, and D. W. Pfaff. 1985. “The Pudendal Nerve-Evoked Response in Axial Muscle.” Experimental Brain Research 61: 175–185.
______. 1987a. “Brainstem Reticular Stimulation Facilitates Back Muscle Motoneuronal Responses to Pudendal Nerve Input.” Brain Research 405: 155–158.
______. 1987b. “Effects of Total and Partial Spinal Transections on the Pudendal Nerve-Evoked Response in Rat Lumbar Axial Muscle.” Brain Research 401: 103–112.
Conrad, L. C. A., and D. W. Pfaff. 1975. “Axonal Projections of Medial Preoptic and Anterior Hypothalamic Neurons.” Science 190: 1112–1114.
______. 1976a. “Efferents from Medial Basal Forebrain and Hypothalamus in the Rat. II. An Autoradiographic Study of the Anterior Hypothalamus.” Journal of Comparative Neurology 169 (2): 221–261.
______. 1976b. “Efferents from Medial Basal Forebrain and Hypothalamus in the Rat. I. An Autoradiographic Study of the Medial Preoptic Area.” Journal of Comparative Neurology 169 (2): 185–220.
Cottingham, S. L., P. A. Femano, and D. W. Pfaff. 1987. “Electrical Stimulation of the Midbrain Central Gray Facilitates Reticulospinal Activation of Axial Muscle EMG.” Experimental Neurology 97: 704–724.
______. 1988. “Vestibulospinal and Reticulospinal Interactions in the Activation of Back Muscle EMG in the Rat.” Experimental Brain Research 73 (1): 198–208.
Cottingham, S. L., and D. W. Pfaff. 1987. “Electrical Stimulation of the Midbrain Central Gray Facilitates Lateral Vestibulospinal Activation of Back Muscle EMG in the Rat.” Brain Research 421: 397–400.
Dupré, C., M. Lovett-Barron, D. W. Pfaff, and L.-M. Kow. 2010. “Histaminergic Responses by Hypothalamic Neurons That Regulate Lordosis and Their Modulation by Estradiol.” Proceedings of the National Academy of Sciences of the United States of America 107 (27): 12311–12316.
Femano, P. A., S. Schwartz-Giblin, and D. W. Pfaff. 1984a. “Brain Stem Reticular Influences on Lumbar Axial Muscle Activity. I. Effective Sites.” American Journal of Physiology 246: R389–R395.
______. 1984b. “Brain Stem Reticular Influences on Lumbar Axial Muscle Activity. II. Temporal Aspects.” American Journal of Physiology 46: R396–R401.
Kow, L.-M., M. O. Montgomery, and D. W. Pfaff. 1977. “Effects of Spinal Cord Transections on Lordosis Reflex in Female Rats.” Brain Research 123: 75–88.
______. 1979. “Triggering of Lordosis Reflex in Female Rats with Somatosensory Stimulation: Quantitative Determination of Stimulus Parameters.” Journal of Neurophysiology 42: 195–202.
Kow, L.-M., S. Pataky, C. Dupré, A. Phan, N. Martín-Alguacil, and D. W. Pfaff. 2016. “Analyses of Rapid Estrogen Actions on Rat Ventromedial Hypothalamic Neurons.” Steroids 111: 100–112.
Kow, L.-M., and D. W. Pfaff. 1973. “Effects of Estrogen Treatment on the Size of Receptive Field and Response Threshold of Pudendal Nerve in the Female Rat.” Neuroendocrinology 13: 299–313.
______. 1975. “Dorsal Root Recording Relevant for Mating Reflexes in Female Rats: Identification of Receptive Fields and Effects of Peripheral Denervation.” Journal of Neurobiology 6: 23–37.
______. 1976. “Sensory Requirements for the Lordosis Reflex in Female Rats.” Brain Research 101: 47–66.
______. 1979. “Responses of Single Units in Sixth Lumbar Dorsal Root Ganglion of Female Rats to Mechanostimulation Relevant for Lordosis Reflex.” Journal of Neurophysiology 42: 203–213.
______. 1982. “Responses of Medullary Reticulospinal and Other Reticular Neurons to Somatosensory and Brainstem Stimulation in Anesthetized or Freely-Moving Ovariectomized Rats with or without Estrogen Treatment.” Experimental Brain Research 47: 191–202.
______. 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.
______. 1987. “Responses of Ventromedial Hypothalamic Neurons in Vitro to Norepinephrine: Dependence on Dose and Receptor Type.” Brain Research 413 (2): 220–228.
______. 1995. “Functional Analyses of α1-Adrenoceptor Subtypes in Rat Hypothalamic Ventromedial Nucleus Neurons.” European Journal of Pharmacology 282: 199–206.
Kow, L.-M., Y.-F. Tsai, N. G. Weiland, B. S. McEwen, and D. W. Pfaff. 1995. “In Vitro Electro-Pharmacological and Autoradiographic Analyses of Muscarinic Receptor Subtypes in Rat Hypothalamic Ventromedial Nucleus: Implications for Cholinergic Regulation of Lordosis.” Brain Research 694: 29–39.
Kow, L.-M., G. D. Weesner, and D. W. Pfaff. 1992. “Adrenergic Agonists Act on Ventromedial Hypothalamic α-Receptors to Cause Neuronal Excitation and Lordosis Facilitation: Electrophysiological and Behavioral Evidence.” Brain Research 588: 237–245.
Kow, L.-M., F. P. Zemlan, and D. W. Pfaff. 1980. “Responses of Lumbosacral Spinal Units to Mechanical Stimuli Related to Analysis of Lordosis Reflex in Female Rats.” Journal of Neurophysiology 43: 27–45.
Krieger, M. S., L. C. A. Conrad, and D. W. Pfaff. 1979. “An Autoradiographic Study of the Efferent Connections of the Ventromedial Nucleus of the Hypothalamus.” Journal of Comparative Neurology 183: 785–816.
Lee, A. W., A.
Kyrozis, V. Chevaleyre, L. M. Kow, N. Devidze, Q. Zhang, A. M. Etgen, and D. W. Pfaff. 2008. “Estradiol Modulation of Phenylephrine-Induced Excitatory Responses in Ventromedial Hypothalamic Neurons of Female Rats.” Proceedings of the National Academy of Sciences of the United States of America 105 (20): 7333–7338.
Manogue, K., L.-M. Kow, and D. W. Pfaff. 1980. “Selective Brain Stem Transections Affecting Reproductive Behavior of Female Rats: The Role of Hypothalamic Output to the Midbrain.” Hormones and Behavior 14: 277–302.
Modianos, D. T., and D. W. Pfaff. 1976. “Brain Stem and Cerebellar Lesions in Female Rats. II. Lordosis Reflex.” Brain Research 106: 47–56.
______. 1977. “Facilitation of the Lordosis Reflex in Female Rats by Electrical Stimulation of the Lateral Vestibular Nucleus.” Brain Research 134: 333–345.
______. 1979. “Medullary Reticular Formation Lesions and Lordosis Reflex in Female Rats.” Brain Research 171: 334–338.
Morrell, J. I., and D. W. Pfaff. 1982. “Characterization of Estrogen-Concentrating Hypothalamic Neurons by Their Axonal Projections.” Science 217: 1273–1276.
______. 1983. “Retrograde HRP Identification of Neurons in the Rhombencephalon and Spinal Cord of the Rat That Project to the Dorsal Mesencephalon.” American Journal of Anatomy 167: 229–240.
Morrell, J. I., T. D. Wolinsky, M. S. Krieger, and D. W. Pfaff. 1982. “Autoradiographic Identification of Estradiol-Concentrating Cells in the Spinal Cord of the Female Rat.” Experimental Brain Research 45: 144–150.
Pfaff, D. W. 1980. Estrogens and Brain Function. Heidelberg: Springer.
Pfaff, D. W., C. Diakow, M. Montgomery, and F. A. Jenkins. 1978. “X-ray Cinematographic Analysis of Lordosis in Female Rats.” Journal of Comparative and Physiological Psychology 92 (5): 937–941.
Pfaff, D. W., A. Korotzer, S. Schwartz-Giblin, and S. L. Cottingham. 1990. “Hypothalamic Effects on Medullary Reticular Activation of Deep Back Muscle EMG.” Physiology and Behavior 47 (1): 185–196.
Pfaff, D. W., and C. Lewis. 1974. “Film Analyses of Lordosis in Female Rats.” Hormones and Behavior 5 (4): 317–335.
Pfaff, D. W., M. Montgomery, and C. Lewis. 1977. “Somatosensory Determinants of Lordosis in Female Rats: Behavioral Definition of the Estrogen Effect.” Journal of Comparative and Physiological Psychology 91: 134–145.
Pfaff, D. W., and Y. Sakuma. 1979a. “Deficit in the Lordosis Reflex of Female Rats Caused by Lesions in the Ventromedial Nucleus of the Hypothalamus.” Journal of Physiology 288: 203–210.
______. 1979b. “Facilitation of the Lordosis Reflex of Female Rats from the Ventromedial Nucleus of the Hypothalamus.” Journal of Physiology 288: 189–202.
Pfaff, D. W., and S. Schwartz-Giblin. 1988. “Cellular Mechanisms of Female Reproductive Behaviors.” In The Physiology of Reproduction. Edited by E. Knobil and J. Neill. New York: Raven, chapter 35, 1487–1568.
Robbins, A., D. W. Pfaff, and S. Schwartz-Giblin. 1992. “Reticulospinal and Reticuloreticular Pathways for Activating the Lumbar Back Muscles in the Rat.” Experimental Brain Research 92: 46–58.
Robbins, A., S. Schwartz-Giblin, and D. W. Pfaff. 1990. “Ascending and Descending Projections to Medullary Reticular Formation Sites Which Activate Deep Lumbar Back Muscles in the Rat.” Experimental Brain Research 80 (3): 463–474.
Sakuma, Y., and D. W. Pfaff. 1979a. “Facilitation of Female Reproductive Behavior from Mesencephalic Central Gray in the Rat.” American Journal of Physiology 237: R278–R284.
______. 1979b. “Mesencephalic Mechanisms for Integration of Female Reproductive Behavior in the Rat.” American Journal of Physiology 237: R285–R290.
______. 1980a. “Cells of Origin of Medullary Projections in Central Gray of Rat Mesencephalon.” Journal of Neurophysiology 44: 1002–1011.
______. 1980b. “Convergent Effects of Lordosis-Relevant Somatosensory and Hypothalamic Influences on Central Gray Cells in the Rat Mesencephalon.” Experimental Neurology 70: 269–281.
______. 1980c. “Excitability of Female Rat Central Gray Cells with Medullary Projections: Changes Produced by Hypothalamic Stimulation and Estrogen Treatment.” Journal of Neurophysiology 44: 1012–1023.
______. 1981. “Electrophysiologic Determination of Projections from Ventromedial Hypothalamus to Midbrain Central Gray: Differences between Female and Male Rats.” Brain Research 225: 184–188.
______. 1982. “Properties of Ventromedial Hypothalamic Neurons with Axons to Midbrain Central Gray.” Experimental Brain Research 46: 292–300.
Schwartz-Giblin, S., P. Femano, and D. W. Pfaff. 1984. “Axial Electromyogram and Intervertebral Length Gauge Responses during Lordosis Behavior in Rats.” Experimental Neurology 85: 297–315.
Schwartz-Giblin, S., M. Halpern, and D. W. Pfaff. 1984. “Segmental Organization of Rat Lateral Longissimus, a Muscle Involved in Lordosis Behavior: EMG and Muscle Nerve Recordings.” Brain Research 299: 247–257.
Schwartz-Giblin, S., and D. W. Pfaff. 1980. “Implanted Strain Gauge and EMG Amplifier to Record Motor Behavior in Unrestrained Rats.” Physiology and Behavior 25: 475–479.
______. 1990. “Ipsilateral and Contralateral Effects on Cutaneous Reflexes in a Back Muscle of the Female Rat: Modulation by Steroids Relevant for Reproductive Behavior.” Journal of Neurophysiology 64 (3): 835–846.
Schwartz-Giblin, S., L. Rosello, and D. W. Pfaff. 1983. “A Histochemical Study of Lateral Longissimus Muscle in Rat.” Experimental Neurology 79: 497–518.
Zemlan, F., L.-M. Kow, J. I. Morrell, and D. W. Pfaff. 1979. “Descending Tracts of the Lateral Columns of the Rat Spinal Cord: A Study Using the Horseradish Peroxidase and Silver Impregnation Techniques.” Journal of Anatomy 128: 489–512.
Zemlan, F. P., L.-M. Kow, and D. W. Pfaff. 1983. “Effect of Interruption of Bulbospinal Pathways on Lordosis, Posture, and Locomotion.” Experimental Neurology 81: 177–194.
Zemlan, F. P., and D. W. Pfaff. 1979. “Topographical Organization in Medullary Reticulospinal Systems as Demonstrated by the Horseradish Peroxidase Technique.” Brain Research 174: 161–166.
Zhou, J., A. W. Lee, Q. Zhang, L. M. Kow, and D. W. Pfaff. 2007. “Histamine-Induced Excitatory Responses in Mouse Ventromedial Hypothalamic Neurons: Ionic Mechanisms and Estrogenic Regulation.” Journal of Neurophysiology 98 (6): 3143–3152.
3
HORMONAL REGULATION OF GENE EXPRESSION IN THE BRAIN
Problem: The estrogen receptors studied in Chapter 1 are transcription factors. What does that have to do with brain function and behavior? Is it possible to make strong causal links from molecular chemistry to physiology (ion channels) and, ultimately, to a complete mammalian behavior?
In Chapter 1, I described our early work on hormone receptors in the brain, and in Chapter 2 how they helped in working out the neural circuit for producing a female reproductive behavior. After we worked out that circuit and were entering the era in which we could apply the techniques of molecular biology to the brain, we faced two challenges in discovering how estrogens, as they drive lordosis behavior, activate the expression of specific genes in specific neurons. First, at that time, gene sequences were not as fully described as they are now, so designing accurate molecular probes offered a challenge. Second, we had to devise assays of sufficient sensitivity and spatial resolution to achieve quantitative assays of gene expression in individual neurons. This chapter will show how, when we met these challenges, we drew an entire series of genes into the explanation of a mammalian behavior.
Lucky
I got lucky. The hormone receptors I had discovered in the brain (Chapter 1) turned out to be “ligand-activated transcription factors”—proteins that, when the hormone was bound, would then bind to specific elements of DNA and facilitate the expression of hormone-dependent genes specifically in those neurons. My laboratory was effectively able to walk our scientific questions and problems into the arena of modern molecular biology.
It was incredibly exciting to have the opportunity to link the neural circuitry we discovered (includi
ng specific neurotransmitters and ion channels) and reproductive behaviors to the chemistry of gene transcription. But there were challenges as well. Could we use in situ hybridization in a way that was sensitive enough to study messenger RNA (mRNA) levels in individual neurons? Could we achieve accurate quantification? The answers to both questions turned out to be yes. The reward for sticking with the program, gene after gene, year after year, was that we had the evidence to show, conceptually and factually, how these transcriptional systems overdetermine the occurrence of reproductive behavior and synchronize it with ovulation.
In this chapter, I discuss, first, how we discovered several genes whose expression was turned on in reproductive behavior-controlling neurons by estrogen treatment. Second, we recently found protranscriptional chromatin changes caused by estrogens over the promoters of those genes. Third, in a slightly more sophisticated inquiry, we found interactions among transcription factors, estrogen receptors (ER), and thyroid hormone receptors (TR). All three are discussed here.
Further, molecular changes in behavior-critical hypothalamic neurons were accompanied by morphological alterations associated with higher levels of synthetic activity. In this chapter, this will be discussed last.
Reproductive Behavior Requires Hormone-Dependent Synthetic Events in Hypothalamic Neurons
Even with a lot of neuroanatomy, histochemistry, and electrophysiology behind me, I was scared to face the question of estrogen-stimulated newly synthesized proteins in the brain. My only background comprised courses in organic chemistry at Harvard and biochemistry at the Massachusetts Institute of Technology (MIT). I marshaled my courage to ask advice of Rockefeller University professor Stanford Moore, a famous protein chemist. Moore and his Rockefeller colleague William Stein had won the Nobel Prize for chemistry based on their first-ever determination of the amino acid sequence of an enzyme (RNAse). Thus, it was especially kind of Professor Moore to invite me to lunch in the Rockefeller dining room and explain the technique of protein chemical discovery that would be most likely to work in small amounts of hypothalamic tissue. More than that, he guaranteed the leadership of his most experienced laboratory member, Peter Blackburn, to help make this project succeed.
