by Donald Pfaff
Figure 8.3. The vast axonal distributions ascending and descending from neurons in the medullary reticular formation nucleus gigantocellularis (NGC) groups. This widespread influence accounts for the NGC’s influence on a range of behaviors, including but not limited to lordosis behavior. (Adapted from Pfaff, Martin, and Faber 2012.)
Reciprocal Links between Generalized Arousal and Sexual Arousal
We consider the sexual behaviors we have explained as requiring a heightened state of CNS arousal. In linking this heightened state to sexual mechanisms, it was found that the heightened state of GA can be abolished by a mutation of the estrogen receptor α (ER-α) gene (Garey et al. 2003). In search of an overarching theoretical formulation, pediatric surgeon Justine Schober in my laboratory envisioned bidirectional arousal mechanisms: GA heightening sexual arousal and vice versa (Schober, Weil, and Pfaff 2011). Of course, we pursued the estrogenic mechanisms.
For example, estrogens can heighten CNS arousal by reducing the expression of a sleep-active system (Ribeiro, Pfaff, and Devidze 2009). We had previously found that estrogens reduce lipocalin-type prostaglandin D synthase (L-PGDS) expression in a region-dependent manner in the mouse preoptic area (POA). This result linked sex hormones with sleep-wake cycle regulation. In turn, the somnogenic effects of prostaglandin D2 have been shown to be mediated by increases in adenosine, and a select group of sleep-active ventrolateral preoptic nucleus neurons are directly activated by adenosine 2A agonists. We hypothesized that increased arousal after estrogen administration is mediated by a reduction of L-PGDS and lowered adenosine 2A receptor expression in the POA.
To test this hypothesis, we studied the running-wheel activity of ovariectomized female mice treated with oil or different doses of estradiol benzoate (EB) (Ribeiro, Pfaff, and Devidze 2009). This was followed by quantitative reverse-transcriptase polymerase chain reaction analysis to determine the mRNA expression of genes related to sleep and arousal in brain region extracts from oil-treated and EB-treated mice. The running-wheel activity was increased in estrogen-treated mice, and these effects followed an inverted U dose–response curve. The most effective dose (1.25 micrograms of EB per capsule) increased running-wheel activity more than 2.5-fold as compared with the control animals, and EB doses that were higher or lower were less effective. Increases in running-wheel activity were accompanied by decreased L-PGDS mRNA in the POA and decreased adenosine 2A receptor mRNA in the POA and ventrolateral preoptic nucleus. Given that EB-treated animals have higher motor activity and lower levels of L-PGDS and adenosine 2A receptor mRNA in sleep-active areas, our findings supported the hypothesis that EB may increase behavioral arousal by decreasing the levels of well-known sleep-inducing molecules within the preoptic region.
Another behavior that depends on a heightened state of arousal, in addition to sex, is fear. Estrogens have been shown to affect nonreproductive behaviors in humans and rodents, including “anxiety,” fear, and activity levels. We examined the effect of EB in ovariectomized mice on a range of behavioral paradigms that measured anxiety (open field, dark–light transition, elevated plus maze), activity (running wheel), or conditioned fear learning (Morgan and Pfaff 2001). In open field conditions, the animals receiving the vehicle treatment spent more time in the center than the EB-treated animals and were more active overall. In the dark–light transition environment, the animals receiving the vehicle treatment were more active than the EB-treated animals in both the dark and light compartments and made more transitions between the two. In the elevated plus maze environment, animals receiving the vehicle treatment entered a greater number of arms. During conditioned fear learning, the EB-treated animals froze more than the vehicle-treated animals when exposed to the conditioned stimulus. In contrast, with the home cage running wheel, the EB-treated animals were more active than those receiving the vehicle treatment.
Factor analysis was used to characterize the intertask correlations of females’ behavior and to explore the possibility that estrogen may have an impact on a GA factor. In sum, estrogen treatment heightened the fear responses in a range of fear- and anxiety-provoking situations, while increasing activity in the safer environment of the running wheel. Our results suggested, therefore, that EB treatments resulted in a generally more aroused animal: a higher GA.
A few years later, we used a seminatural environment to investigate the role of ER-α in the VMH, the POA, the medial amygdala, and the bed nucleus of stria terminalis (BNST) in sociosexual behavior in female rats (Snoeren et al. 2015). We conducted two sets of experiments, with the VMH and POA investigated in the first set, and the medial amygdala and BNST in the second set. The VMH and POA receive intense projections from the medial amygdala and BNST. We used a short-hairpin RNA encoded within an adeno-associated viral vector directed against the gene for ER-α to reduce the number of neurons expressing ER-α in the VMH or POA (first set of experiments) or in the BNST or medial amygdala (second set of experiments) in female rats. The rats were housed in groups of four ovariectomized females and three males in a seminatural environment for 8 days.
Compared with traditional test setups, the seminatural environment provided an arena in which the rats could express their full behavioral repertoire, which allowed us to investigate multiple aspects of social and sexual behaviors in groups. Behavioral observation was performed after estrogen and progesterone injections. Most important for my point here, a reduction of ER-α expression in the VMH or POA diminished the display of courtship behaviors and lordosis responses compared with the control rats. The loss of the high-energy behaviors involved in the females’ courtship indicated a reduction of GA in females, for whom ER-α expression was substantially reduced in the VMH or POA. In all three experiments, it appeared that estrogens working through ER-α heightened GA.
Principle inferred: Generalized central nervous system arousal exists and supports sex arousal and subsequent sexual behavior.
Further Reading
Calderon, D. P., M. Kilinc, A. Maritan, J. R. Banavar, and D. W. Pfaff. 2016. “Generalized CNS Arousal: An Elementary Force within the Vertebrate Nervous System.” Neuroscience and Biobehavioral Reviews 68: 167–176.
Calderon, D. P., A. Proekt, and D. W. Pfaff. 2016. “Modulation of Nucleus Gigantocellularis Neurons Regulate Cortical Arousal.” Nature Neuroscience (under review).
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.
Easton, A., E. Dwyer E, and D. W. Pfaff. 2006. “Estradiol and Orexin-2 Saporin Actions on Multiple Forms of Behavioral Arousal in Female Mice.” Behavioral Neuroscience 120 (1): 1–9.
Garey, J., A. Goodwillie, J. Frohlich, M. Morgan, J.-A. Gustafsson, O. Smithies, K. S. Korach, S. Ogawa, and D. W. Pfaff. 2003. “Genetic Contributions to Generalized Arousal of Brain and Behavior.” Proceedings of the National Academy of Sciences of the United States of America 100 (19): 11019–11022.
Garey, J., L.-M. Kow, W. Huynh, and D. W. Pfaff. 2002. “Temporal and Spatial Quantitation of Reproductive Behaviors among Mice Housed in a Semi-Natural Environment.” Hormones and Behavior 42 (3): 294–306.
Hudson, A. E., D. P. Calderon, D. W. Pfaff, and A. Proekt. 2014. “Recovery of Consciousness Is Mediated by a Network of Discrete Metastable Activity States.” Proceedings of the National Academy of Sciences of the United States of America 111 (25): 9283–9288.
Keenan, D. M., A. W. Quinkert, and D. W. Pfaff. 2015. “Stochastic Modeling of Mouse Motor Activity under Deep Brain Stimulation: The Extraction of Arousal Information.” PLoS Computational Biology 11 (2): e1003883.
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): 1–10.
______. 1986. “Vasopressin Excites
Ventromedial Hypothalamic Glucose-Responsive Neurons in Vitro.” Physiology and Behavior 37: 153–158.
______. 1987. “Responses of Ventromedial Hypothalamic Neurons in Vitro to Norepinephrine; Dependence on Dose and Receptor Type.” Brain Research 413: 220–228.
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.
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.
Martin, E. M., N. Devidze, D. N. Shelley, L. Westberg, C. Fontaine, and D. W. Pfaff. 2011. “Molecular and Neuroanatomical Characterization of Single Neurons in the Mouse Medullary Gigantocellular Reticular Neurons.” Journal of Comparative Neurology 519 (13): 2574–2593.
Morgan, M. A., and D. W. Pfaff. 2001. “Effects of Estrogen on Activity and Fear-Related Behaviors in Mice.” Hormones and Behavior 40 (4): 472–482.
Pfaff, D. W. 1980. Estrogens and Brain Function. Heidelberg: Springer.
______. 2006. Brain Arousal and Information Theory. Cambridge, MA: Harvard University Press.
Pfaff, D. W., and J. R. Banavar. 2007. “A Theoretical Framework for CNS Arousal.” BioEssays 29 (8): 803–810.
Pfaff, D. W., E. M. Martin, and D. Faber. 2012. “Origins of Arousal: Roles for Medullary Reticular Neurons.” Trends in Neuroscience 35 (8): 468–476.
Pfaff, D. W., A. C. Ribeiro, J. Matthews, and L.-M. Kow. 2008. “Concepts and Mechanisms of Generalized Central Nervous System Arousal.” Annals of the New York Academy of Sciences 1129 (1): 11–25.
Proekt, A., J. R. Banavar, A. Maritan, and D. W. Pfaff. 2012. “Scale Invariance in the Dynamics of Spontaneous Behavior.” Proceedings of the National Academy of Sciences of the United States of America 109 (26): 10564–10569.
Quinkert, V. V., Z. M. Weil, G. N. Reeke, N. D. Schiff, J. R. Banavar, and D. W. Pfaff. 2011. “Quantitative Descriptions of Generalized Arousal, an Elementary Function of the Vertebrate Brain.” Proceedings of the National Academy of Sciences of the United States of America 108 (Suppl. 3): 15617–15623.
Quiñones-Jenab, V., C. Zhang, S. Jenab, H. E. Brown, and D. W. Pfaff. 1996. “Anesthesia during Hormone Administration Abolishes the Estrogen Induction of Preproenkephalin mRNA in Ventromedial Hypothalamus of Female Rats.” Molecular Brain Research 35 (1–2): 297–303.
Ribeiro, A. C., D. W. Pfaff, and N. Devidze. 2009. “Estradiol Modulates Behavioral Arousal and Induces Changes in Gene Expression Profiles in Brain Regions Involved in the Control of Vigilance.” European Journal of Neuroscience 29 (4): 795–801.
Schober, J., Z. Weil, and D. W. Pfaff. 2011. “How Generalized CNS Arousal Strengthens Sexual Arousal (and Vice Versa).” Hormones and Behavior 59 (5): 689–695.
Snoeren E. M., E. Antonio-Cabrera, T. Spiteri, S. Musatov, S. Ogawa, D. W. Pfaff, and A. Ågmo. 2015. “Role of Oestrogen α Receptors in Sociosexual Behaviour in Female Rats Housed in a Seminatural Environment.” Journal of Neuroendocrinology 27: 803–818.
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.
Valverde, F. 1961. “Reticular Formation of the Pons and Medulla Oblongata. A Golgi Study.” Journal of Comparative Neurology 116 (1): 71–99.
Weil, Z. M., Q. Zhang, A. Hornung, D. Blizard, and D. W. Pfaff. 2010. “Impact of Generalized Brain Arousal on Sexual Behavior.” Proceedings of the National Academy of Sciences of the United States of America 107: 2265–2270.
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: 3143–3152.
9
SEX DIFFERENCE
Problem: To distinguish large, reliable, physiologically important sex differences in behavior from stories that are unreliable or unimportant. Numerous studies have yielded to the temptation to proclaim sex differences in this or that behavior. In truth, the farther a given brain function is from the nitty-gritty—the central phenomena of reproductive (neuro)biology—the less impressive is the cognate sex difference. One exception is the spectrum of behavioral changes collectively called autism.
A single chapter in a scientific account such as this, recounting the experience of a single laboratory, cannot do justice to the rich field of scholarship dealing with sex differences in brain and behavior. With respect to the unique ability of the female brain and pituitary to manage ovulation, the endocrine expertise of Jon Levine at the University of Wisconsin and George Fink at Monash University in Australia covers the field (Fink et al. 2012). The sophisticated genetics of sex differences in behavior have been pioneered by the accomplished behavioral biologist Arthur Arnold at the University of California–Los Angeles, with the corresponding neurochemistry explored aggressively by Margaret McCarthy at the University of Maryland’s School of Medicine. Nevertheless, the work from our laboratory on sex differences links directly to the molecular biology of Chapters 3 and 4 and thus will be covered here.
The central fact is that, among laboratory animals, females do lordosis behavior and males generally do not. In one of our data sets (Pfaff and Zigmond 1971), after estrogen treatment, females showed lordosis thirty-eight times as frequently as males. After estrogen supplemented by progesterone, they showed it thirty-one times as frequently as males.
The major driver of this behavioral sex difference is the concentration of testosterone (and testosterone metabolites) during the final few hours before birth and the first few hours after birth. In the same 1971 study, I included behavioral measurements of groups of males that had been castrated (deprived of testosterone and its metabolites) within hours of birth. Also groups of females that had been injected with testosterone within hours of birth. After estrogen treatment, the neonatally castrated males showed lordosis thirty-seven times as frequently as the males who had intact testes until their adulthood. The control females showed lordosis thirty-eight times as frequently as the females treated neonatally with testosterone. These massive neonatal treatment effects were replicated and extended by experimental conditions that used estrogen supplemented by progesterone (Pfaff and Zigmond 1971).
Mechanisms
As previously mentioned, Margaret McCarthy has conceived of many of the possible routes to sexual differentiation, starting from the pioneering work of Charles Phoenix (with monkeys) and Roger Gorsky (with rats). For McCarthy, the routes boil down to sex differences in cell birth, death, or migration. Obvious examples include the production in the female of a larger nucleus called the anteroventral periventricular nucleus of the hypothalamus (which is crucial for ovulation), and the production in the male of a large “sexually dimorphic nucleus” (which participates in male sexual behavior). Subsequent to the causal routes reviewed by McCarthy, the sexual differentiation of certain neural circuits can be discerned: dendritic elaboration, axonal projection, and synapse formation.
From microarray studies, it is clear that there are sex differences in gene expression in the brain during the critical period of sexual differentiation (Gagnidze, Pfaff, and Mong 2010). We sacrificed mice late on the day of birth (8 to 12 hours after birth), late enough to take advantage of the perinatal peak of testosterone sensitivity but soon enough that sexually differentiated behaviors would not affect gene expression. Surprisingly, 8 percent of genes were differentially expressed in the preoptic area, between neonatal male and female mice. In the medial basal hypothalamus t
he proportion of differentially expressed genes was smaller, 0.9 percent of the 11,000 measured. Many different classes of gene products were represented, from transcription factors to translation factors, metabolic enzymes G proteins, growth factors, kinases, phosphatases cytoskeletal proteins, and synapse-associated proteins. Follow-up studies will examine the results histochemically, gene by gene.
Extensive DNA sequencing during the last 30 years has resulted in knowledge of gene and promoter structures, knowledge that has yielded a molecular neuropharmacology of behavioral sex differences. Most exquisitely, the synthesis of small-interfering RNA (siRNA) and the use of homologous recombination to make gene knockouts have provided tools for the analysis of female sex behavior and maternal behavior, and male sex behavior and aggression.
Female-specific sex behavior (i.e., lordosis behavior) depends on normal expression of the gene encoding the ligand-activated transcription factor estrogen receptor α (ER-α) (Ogawa et al. 1998). Particularly important is the expression in the ventromedial nucleus of the hypothalamus (Musatov et al. 2006). This may be, in part, because of the sexually differentiated effect of estrogens on the expression of the gene for the progesterone receptor (PR) in the ventromedial hypothalamus (Romano et al. 1989). Moreover, antisense DNA against PR messenger RNA (mRNA) microinjected in the ventromedial hypothalamus significantly reduces estrogen-stimulated lordosis behavior (Ogawa et al. 1994). In fact, disruption of the gene for ER-α effectively reversed sex roles in female mice; it caused the females to lose their normal female-typical behavior and to behave and be treated more like males (Ogawa et al. 1996).