by Donald Pfaff
Neurotransmitters and neuropeptides. The fundamental chemistries of neurotransmitters and neuropeptides have by and large remained the same as we move from the animal brain to the human brain.
Neurophysiology. Mechanisms of ion flow and signaling by action potentials and local field potentials are so fundamentally similar among nerve cells of many different species as to preclude any reason to believe that there are major differences between laboratory mammals and humans.
Neuropharmacology. Because the basic facts of neurochemistry and electrophysiology have been conserved, one starts with the supposition that neuropharmacological mechanisms remain similar among laboratory mammals and humans, although that does not mean that developing successful psychopharmacological drugs is easy.
Thus, in so many respects the neural mechanisms I deal with in laboratory animals have been strongly conserved in the human brain.
Lessons inferred: The main message of this chapter is that our comprehensive knowledge of a set of brain / behavioral mechanisms extends to the nervous system the story of how body-wide orchestration permits mammalian reproduction to occur. Several principles emerge: 1) a chain of reproductive behaviors and 2) genes that are hormone-sensitive, stimulating and organizing an entire epoch of instinctive behaviors necessary for reproduction. Because of a high degree of conservation of these mechanisms, our work also explains the physiological aspect of libido. Animals and people do not have to learn these behaviors—they are the kinds of natural behaviors that ethologists studied so famously years ago.
I have used the phrase “hormone-dependent behavioral funnel” to describe the series of courtship and other social behaviors that funnel reproductive competent conspecifics, male and female (and only those), into the same space at the same time. Is the following extrapolation reasonable? The thinking and work of anthropologists such as Sarah Hrdy at the University of California–Davis and ethologists such as Barry Keverne at the University of Cambridge have argued that the expansion of behavioral repertoires from the sorts of systems I have explained will eventually be seen to form the basis of prosocial behaviors that maintain peaceful societies. For those disciplines, such interesting extrapolations may provide the next steps of interesting inquiries.
Further Reading
Albertini, D. F. 2015. “The Mammalian Oocyte.” In Knobil and Neill’s Physiology of Reproduction. 4th ed. 2 vols. Edited by T. M. Plant and A. J. Zeleznik. Amsterdam: Elsevier, 1:59–97.
Anderson, S. M., P. S. MacLean, J. L. McManaman, and M. C. Neville. 2015. “Lactation and Its Hormonal Control.” In Knobil and Neill’s Physiology of Reproduction. 4th ed. 2 vols. Edited by T. M. Plant and A. J. Zeleznik. Amsterdam: Elsevier, 2:2055–2105.
Binder, A. K., W. Winuthayanon, S. C. Hewitt, J. F. Couse, and K. S. Korach. 2015. “Steroid Receptors in the Uterus and Ovary.” In Knobil and Neill’s Physiology of Reproduction. 4th ed. 2 vols. Edited by T. M. Plant and A. J. Zeleznik. Amsterdam: Elsevier, 1:1099–1193.
Fahrbach, S. E., R. L. Meisel, and D. W. Pfaff. 1985. “Preoptic Implants of Estradiol Increase Wheel Running but Not the Open Field Activity of Female Rats.” Physiology and Behavior 35: 985–992.
Fahrbach, S. E., J. I. Morrell, and D. W. Pfaff. 1986. “Effect of Varying the Duration of Pre-test Cage Habituation on Oxytocin Induction of Short-Latency Maternal Behavior.” Physiology and Behavior 37 (1): 135–139.
Fink, G., D. W. Pfaff, and J. Levine. 2012. Handbook of Neuroendocrinology. San Diego: Academic Press / Elsevier.
Lomniczi, A., J. M. Castellano, H. Wright, B. Selcuk, K. Sonmez, and S. R. Ojeda. 2015. “Gene Networks, Epigenetics and the Control of Female Puberty.” In Brain Crosstalk in Puberty and Adolescence. Edited by J.-P. Bourguignon, J.-C. Carel, and Y. Christen. Berlin: Springer, 97–119.
Martín-Alguacil, N., D. W. Pfaff, D. N. Shelley, and J. M. Shober. 2008. “Clitoral Sexual Arousal: An Immunocytochemical and Innervation Study of the Clitoris.” BJU International 101: 1407–1413.
Martín-Alguacil, N., J. M. Schober, L. M. Kow, and D. W. Pfaff. 2006. “Arousing Properties of the Vulvar Epithelium.” Journal of Urology 176 (2): 456–462.
______. 2008a. “Estrogen Receptor Expression and Neuronal Nitric Oxide Synthase in the Clitoris and Preputial Gland Structures of Mice.” BJU International 102: 1719–1723.
Martin-Alguacíl, N., J. M. Schober, D. R. Sengelaub, D. W. Pfaff, and D. N. Shelley. 2008b. “Clitoral Sexual Arousal: Neuronal Tracing Study from the Clitoris through the Spinal Tracts.” Journal of Urology 180: 1241–1248.
McCarthy, M. M., S. R. Chung, S. Ogawa, L.-M. Kow, and D. W. Pfaff. 1991. “Behavioral Effects of Oxytocin: Is There a Unifying Principle?” In Vasopressin. Edited by S. Jard and R. Jamison. Paris: Colloque INSERM / John Libbey Eurotext, 195–212.
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 (1): 230–239.
Pfaff, D. W. 1965. “Cerebral Implantation and Autoradiographic Studies of Sex Hormones.” In Sex Research: New Developments. Edited by J. Money. New York: Holt, Rinehart & Winston, 219–234.
______. 1968. “Autoradiographic Localization of Radioactivity in Rat Brain after Injection of Tritiated Sex Hormones.” Science 161: 1355–1356.
______. 1999. Drive: Neurobiological and Molecular Mechanisms of Sexual Motivation. Cambridge, MA: MIT Press.
Pfaff, D. W., I. Phillips, and R. Rubin. 2004. Principles of Hormone / Behavior Relations. San Diego: Academic Press / Elsevier.
Plant, T., and A. Zeleznik. 2015. Physiology of Reproduction. 4th ed., 2 vols. San Diego: Academic Press / Elsevier.
Ribeiro, A., S. Musatov, 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.
Schober, J., N. Aardsma, L. Mayoglou, D. W. Pfaff, and N. Martín-Alguacil. 2015. “Terminal Innervation of Female Genitalia, Cutaneous Sensory Receptors of the Epithelium of the Labia Minora.” Clinical Anatomy 28: 392–398.
Shubin, N. 2008. Your Inner Fish: A Journey into the 3.5-Billion-Year History of the Human Body. New York: Vintage.
Spiteri, T., S. Musatov, S. Ogawa, A. Ribeiro, D. W. Pfaff, and A. Ågmo. 2010. “Estrogen-Induced Sexual Incentive Motivation, Proceptivity and Receptivity Depend on a Functional Estrogen Receptor Alpha in the Ventromedial Nucleus of the Hypothalamus but Not in the Amygdala.” Neuroendocrinology 91: 142–154.
Spiteri, T., S. Ogawa, S. Musatov, D. W. Pfaff, and A. Ågmo. 2012. “The Role of the Estrogen Receptor α in the Medial Preoptic Area in Sexual Incentive Motivation, Proceptivity and Receptivity, Anxiety, and Wheel Running in Female Rats.” Behavioral Brain Research 230: 11–20.
Tena-Sempere, M. 2015. “Neuroendocrine and Molecular Mechanisms for the Metabolic Control of Puberty: Recent Developments.” In Brain Crosstalk in Puberty and Adolescence. Edited by J.-P. Bourguignon, J.-C. Carel, and Y. Christen. Berlin: Springer, 121–140.
8
CENTRAL NERVOUS SYSTEM AROUSAL FUELING INSTINCTIVE BEHAVIORS
Problem: Underlying all of the behaviors discussed in previous chapters is the most powerful and essential force in the central nervous system: generalized arousal. We need to know how it works.
In both sexes, generalized arousal (GA) potentiates reproductive behaviors. In females, we know exactly how: depolarizing actions of arousal-related neurotransmitters on ventromedial hypothalamic neurons. We have analyzed these phenomena from ion channels through physiology to behavior. These experiments show how a global brain function affects a specific instinctive behavior.
First I discuss data showing that generalized central nervous system (CNS) arousal is required for normal lordosis behavior. I then delve into the theory and supporting data regarding GA itself. Finally, I discuss the linkage between the t
wo forms of arousal: GA and sexual arousal.
Generalized Arousal Required for Reproductive Behavior
Analyzing the behaviors themselves, it became clear to us that courtship and lordosis behaviors could not be performed without a high degree of CNS arousal (Pfaff 1980; Garey et al. 2002). The “courtship” behaviors of female laboratory rats and mice feature very rapid hopping movements, sudden in their initiation and in their halt. This unusual, intense form of locomotion has biological utility in that the male, having sniffed at the rear of the female will be following from the rear and, upon the female’s sudden halt, will bump into her in the proper position for mounting. Further, during the mounts, the female may be supporting the entire weight of the male—in stop-action films we saw the tensely braced female (250 grams) supporting all the weight of a 500-gram male. Then, after lordosis behavior, the female darts quickly away. In a seminatural environment (Garey et al. 2002), this part of the locomotion was the fastest I have ever seen.
An experiment by Ed Roy at the University of Illinois confirmed what we thought from analyzing the behavior itself. He anesthetized female rats during exposures to estradiol that would normally facilitate lordosis behavior, and in doing so he inhibited the induction of behavioral receptivity. Dropping GA by anesthesia blocked the estrogen effect on the behavior. How does this work?
Lee-Ming Kow and I figured this out when we investigated the responses of neurons in the ventromedial hypothalamus (VMH) to arousal-related transmitters (Figure 8.1) (Kow and Pfaff 2005). Inputs from these transmitters would be essential for maintaining the VMH activity required for translating an estrogen effect into lordosis behavior. During recording from VMH neurons in in vitro brain tissue slices through the hypothalamus, we found excitatory responses to glutamate, acetylcholine, and norepinephrine. The overall resting rates of neuronal activity and responses to the transmitters were very similar to those observed in in vivo studies. Later, Christophe Dupré got the same kind of responses using the arousal transmitter histamine, excitation being mediated by H1 receptors (Dupré et al. 2010). Thus, the essential GA action on lordosis can be explained by these VMH effects of arousal-related neurotransmitters.
Ion channels. As introduced in Chapter 3, norepinephrine uses both L-type calcium channels and reduced conductance through potassium channels (A currents) to signal through Gq proteins, thus to activate phospholipase C and protein kinase C. Glutamate acting through N-methyl-D-aspartate receptors coupled directly to ion channels triggers an inward sodium current. Acetylcholine, acting through muscarinic receptors in the VMH, will excite VMH neurons through the inward flow of sodium ions. Histamine, acting through H1 receptors, works through the inhibition of a potassium leak current to depolarize VMH neurons.
Figure 8.1. Left: Estradiol benzoate (E2) treatment increased histamine-induced change of membrane potential and inward current in ventromedial hypothalamus (VMH) neurons. Here see samples of continuous current-clamp recording of VMH neurons from oil-control and E2-treated animals. Holding potential was −55 mV. Neuron from E2-treated animal showed larger depolarization and more action potentials compared with neuron from oil-treated, control animal. Right: Current-voltage (I–V) relationships of peak high-voltage-activated (HVA) barium currents before (Pre), during (PHEN), and after (Post) phenylephrine application to VMH neurons Recordings were obtained using the gramicidin perforated-patch configuration. Phenylephrine increased the depolarizing currents in response to voltage steps. (Adapted from Zhou et al. 2007; and Lee et al. 2008.)
Likewise, we were able to show that anesthesia disrupts one of the transcriptional events that underlie estrogenic actions in the VMH to promote lordosis behavior (Quiñones-Jenab et al. 1996). By quantitative in situ hybridization and slot-blot analysis techniques, we found a 1.8-fold increase in preproenkephalin (PPE) messenger RNA (mRNA) levels in the VMH after 1 hour of estrogen treatment in ovariectomized female rats. Anesthetizing the rats with pentobarbital for 1 hour during the exposure to estrogen blocked the estrogen induction of PPE mRNA in the VMH. We saw a similar trend using a different anesthetic, chloral hydrate. It appears that neuronal activity is required for the early phase of estrogen induction of PPE mRNA levels in the VMH. Generalized CNS arousal, manifested in arousal-related transmitter inputs to the VMH, is required for lordosis and for lordosis-related gene expression.
A similar conclusion holds true for male sex behavior. When he was in our laboratory, Zachary Weil showed that males bred for high GA were extremely quick to mount the females and would also ejaculate easily; males bred for low GA were extremely sluggish, took a long time to approach the females, and were slow to ejaculate (Weil et al. 2010). Here as well, high GA is necessary to support sex behavior.
Generalized Central Nervous System Arousal: Theory and Mechanisms
To account for the ability of the vertebrate brain to regulate high-stakes behaviors such as sex and fear, as well as the ability of the vertebrate brain to trigger behaviors quickly in emergency situations, we devised the concept of “generalized CNS arousal” (Pfaff 2006; Pfaff et al. 2008). The GA systems include the following operating requirements. 1) GA systems have to be labile, trigger-quick and not sluggish, and must be sensitive to the momentary state of the organism. 2) On the one hand, convergence is required; all sensory stimuli activate the same set of arousal subsystems, which, in turn, support each other. 3) On the other hand, GA outputs must diverge; they activate cerebral cortex, spinal cord, autonomic nervous systems, and endocrine organs to initiate behavior. 4) Finally, they must be robust—they cannot fail. Survival of the organism depends on adequate CNS arousal.
Further, I adopted the simplest possible operating definition of GA (Pfaff 2006; Pfaff et al. 2008; Quinkert et al. 2011) that leads to physical, quantitative measurements: a more aroused animal or human 1) is more responsive to sensory stimuli in all sensory modalities, 2) emits more voluntary motor activity, and (3) is more reactive emotionally.
GA verifiably exists (as reviewed at length in Pfaff, Martin, and Faber 2012; Calderon, Kilinc, et al. 2016). The evidence for the existence of GA can be sorted broadly into four classes: psychological and ethological (the oldest line of evidence), genetic (the newest), statistical, and mechanistic.
The need for rapid responses in emergency situations and for exquisite sensitivity to small (but important) changes in the environment led us to think about nonlinear dynamics (Pfaff and Banavar 2007). The mathematics of physical systems undergoing a phase transition served us well. For example, mathematician Edward Ott’s “controlled chaotic” phase attracted us in that it is sensitive to the initial state of the CNS, supposes dynamics that are deterministic, and provides tremendous amplification of neuronal activity needed for CNS arousal. The phase transition from nonaroused to aroused generates behaviors described by power laws and results in “dynamic criticality” of the CNS. A resulting phenomenon called “scale invariance” leads to several striking consequences in physical systems. The first is universality: even though the elementary constituents of a system can vary, criticality and scale invariance emerge as the collective behavior of a many-body system. A second consequence of scale invariance is that the dynamics of a system at criticality is characterized by a plethora of time scales—there is no single predictable time scale.
This theoretical approach predicts behavioral results summarized as follows. We presented a general framework for understanding how such scale invariance may arise in nonequilibrium systems, including those that arouse the CNS and account for the initiation of behavior (Proekt et al. 2012). Using prolonged behavioral observations with high temporal resolution that yielded more than 51 million data points, we demonstrated that the predictions of the theoretical framework mentioned above are in agreement with a detailed analysis of mouse behavioral arousal observed in a simple unchanging environment. Power law mathematics fit the behavioral results. Scale invariance observed in the dynamics of behavioral arousal must arise from the dynamics intrinsic to the brain.
Mechanisms Underlying Generalized Arousal
The very fact that neuroanatomists, neurophysiologists, neurochemists, and geneticists have deciphered mechanisms that contribute to the activation of behavior provides further evidence for the existence of GA. We have reviewed these results comprehensively (Pfaff, Martin, and Faber 2012; Calderon, Kilinc, et al. 2016). Briefly, at least six major ascending systems contribute to forebrain arousal, including the noradrenergic, glutamatergic, and cholinergic systems. Medullary reticulospinal systems regulate descending arousal signals, which is important because the operating definition of increased GA includes the measurement of increased motor activity.
Figure 8.2. Nucleus gigantocellularis (NGC) neurons can regulate cortical arousal (see arrows on ascending limbs of bifurcating axons) and the initiation of motor activity (see arrows on descending limbs), thus contributing to reticulospinal pathways. (Adapted from Valverde 1961.)
Clearly, a large number of genes and their products contribute to the regulation of GA. For example, it should first be noted that each neurotransmitter or neuropeptide that contributes to activity changes in GA pathways depends on at least four classes of genes for its proper regulation: genes for synthetic enzymes, genes for receptors, transporter genes, and genes encoding catabolic enzymes.
Optogenetics. A group of very large neurons in the medial medullary reticular formation (Figure 8.2) called the nucleus gigantocellularis (NGC) contributes to both ascending and descending arousal regulation (Figure 8.3). These neurons express vGlut2, indicating that they are glutamatergic neurons, and they express receptors for GA-related transmitters and peptides, including hypocretin and opioid receptors (Martin et al. 2011). Analyses of behavioral data with mathematical statistics have confirmed that, when stimulating the thalamic projections of these NGC neurons, behavioral arousal is activated (Keenan et al. 2015). We have used optogenetics 1) to turn on these glutamatergic neurons and, 2) in separate experiments, to turn off neighboring GABAergic neurons (Calderon, Proekt, and Pfaff 2016). Both optogenetic maneuvers applied among NGC neurons serve to activate high-frequency activity and reduce low-frequency activity as seen in cortical electroencephalograms, even those of deeply anesthetized animals. Mice can be aroused such that they can attempt to walk away even though their heads are fixed stereotactically in space. Our theory is that these are the most important neurons for initiating high levels of GA, and thus to serve sexual arousal in turn.