by Donald Pfaff
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______. 1996. “Gonadotropin-Releasing Hormone Gene Expression in Teleosts.” Brain Research: Molecular Brain Research 41 (1–2): 216–227.
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______. 1973. “Luteinizing Hormone Releasing Factor (LRF) Potentiates Lordosis Behavior in Hypophysectomized Ovariectomized Female Rats.” Science 182: 1148–1149.
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______. 1983. “Modulation of the Lordosis Behavior of Female Rats by LHRH, Its Antiserum and Analogs in the Mesencephalic Central Gray.” Neuroendocrinology 36: 218–224.
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Schwanzel-Fukuda, M., M. S. Garcia, J. I. Morrell, and D. W. Pfaff. 1987. “Distribution of Luteinizing Hormone-Releasing Hormone in the Nervus Terminalis and Brain of the Mouse Detected by Immunocytochemistry.” Journal of Comparative Neurology 255: 231–244.
Schwanzel-Fukuda, M., K. Jorgenson, H. Bergen, G. Weesner, and D. W. Pfaff. 1992. “Biology of Normal LHRH Neurons during and after Their Migration from Olfactory Placode.” Endocrine Reviews 13: 623–633.
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______. 1983b. “Absence of Oestradiol Concentration in Cell Nuclei of LHRH-Immunoreactive Neurons.” Nature 304: 345–347.
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6
NEUROPEPTIDE: OXYTOCIN
Problem: We have solved the mechanisms for a simple social behavior, in this case a sexual behavior. But how do we conceive of sexual behaviors as leading to an understanding of the panoply of prosocial behaviors? A pair of genes are exquisitely sensitive to estrogens: the gene encoding oxytocin, a small 9-amino-acid peptide, and the gene encoding its receptor, the oxytocin receptor. In fact, those two transcriptional inductions could multiply each other’s effects on behavior. These genes are important for female sex behaviors and are also crucial for maternal behaviors. They have been reported to foster many positive social behaviors in experimental animals and humans. Could a single peptide produced in hypothalamic neurons (and its receptor) support sexuality as well as a large number of prosocial behaviors?
Chemistry, Molecular Biology
Oxytocin, a 9-amino-acid peptide (Figure 6.1, top), is produced in a restricted subset of hypothalamic neurons (Rhodes et al. 1981a,b). Most well-known in this regard are the magnocellular neurons in the paraventricular nucleus and supraoptic nucleus, but Rockefeller graduate student Harker Rhodes discovered other, tiny neuronal groups along what appeared to me to be a likely migration path. Supraoptic hypothalamic neurons send axons to the posterior pituitary for deposition into the circulation. That distribution route helps to account for the widespread physiological effects of oxytocin noted herein.
Figure 6.1. The 9-amino-acid neuropeptides oxytocin (top) and vasopressin (bottom) have similar structures but mediate different social behavior responses.
Oxytocin, working through a G-protein coupled receptor, is crucial for stimulating the uterine contractions that permit birth of any newborn mammal; then it stimulates the mammary glands to permit successful feeding of the infant from the breasts. On a more exotic note, some studies have linked oxytocin to sexual arousal in women and to the amelioration of symptoms of autism in young boys.
Rockefeller graduate student Lily Conrad discovered a remarkably wide set of projections from the paraventricular nucleus not only within the hypothalamus—such as to the ventromedial hypothalamus (VMH)—but also to the midbrain and hindbrain (Conrad and Pfaff 1975, 1976a,b). Her findings were echoed and extended by Clif Saper, then at the Washington University School of Medicine (and now at Harvard Medical School). Clif showed projections all the way to the spinal cord.
The chemistry of arginine-vasopressin (AVP; see Figure 6.1, bottom) is very similar to that of oxytocin, a fact that led to an evolutionary analysis of the derivation of both from ancient nonapeptide precursors. AVP works through four receptor subtypes to accomplish a variety of physiological regulations throughout the body. For example, AVP is crucial to the body’s retention of water and for the regulatio
n of blood vessel diameter. These functions are accomplished not only by a complex set of actions in the kidney but also by actions on the blood vessel walls themselves.
The conservation of major features of oxytocinergic systems between laboratory animals and humans also has drawn our attention. To a large extent, the mechanisms and functions of oxytocin that were discovered in mice and rats have been shown to hold true for human physiology as well.
In the areas of the central nervous system (CNS) that are most interesting to me, two paradoxes provide incentives for future studies. On the one hand, Larry Young, a molecular neurobiologist at Emory University, has proven beyond doubt that the neuroanatomical locations and functions of certain AVP receptor subtypes support friendly social behaviors (such as “side by side” affiliation in voles). On the other hand, many researchers, notably including Elliott Albers at Georgia State University, have implicated certain AVP-producing neurons in aggressive behavior. A second paradox is that even though the behavioral functions of AVP and oxytocin are sometimes quite different, in the VMH AVP effectively stimulates oxytocin-responsive neurons (Ragnauth et al. 2004) and in fact has a high affinity for oxytocin receptors (OTR).
A further intriguing molecular detail is that my former collaborator Dietmar Richter, the founder and chief of the molecular neurobiology unit at the University of Hamburg, discovered that not only are the oxytocin and AVP genes near each other on the same chromosome (human, chromosome 20; mouse, chromosome 2), but also that an ancient transposable element, a long interspersed repeated DNA element (LINE) separates them. The function of this LINE element, whose product stays within the nucleus, remains obscure.
I was first attracted to oxytocin because of its simplicity as a nonapeptide and, in particular, its restricted three-dimensional shape enforced by the disulphide bond (see Figure 6.1). Its estrogen sensitivity rendered it compelling, as I now discuss.
Molecular endocrinology. Most important for the story of this book, estrogenic hormones increase the transcription rates from both the oxytocin gene and from the gene for the OTR (Chung et al. 1991; Quinones-Jenab et al. 1997). We have reported on estrogenic effects on OTR-related histone modifications (Figure 6.2) that could contribute to the transcriptional effect (Gagnidze et al. 2013). That was first covered in Chapter 3, where I also pointed out that these two effects could multiply each other in their eventual physiological effects on behavior (Pfaff 1988). I summarize the relevant part of that story here.
Brain, Behavior
With respect to lordosis behavior, we know that regulated gene expression in single neurons can be linked to biophysical events and to lordosis behavior (see Chapter 3). In the case of estrogen-regulated gene expression in neurons in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (the VMH) are crucial. As seen in Chapter 2, these VMH nerve cells are essential for lordosis behavior. In our laboratory, postdoctoral researcher Nino Devidze asked what genes are coexpressed in neurons that have high levels of messenger RNA (mRNA) for estrogen receptors. We were able to isolate and measure certain mRNAs from individual VMH neurons collected from the rat hypothalamus (Devidze et al. 2005). A large number of neurons expressed mRNA for estrogen receptor α (ER-α), and these neurons were not identical with the population of VMH neurons expressing ER-β. An extremely high proportion of neurons expressing ER also coexpressed mRNA for OTR. This fact matched the known participation of oxytocin binding and signaling in sexual and affiliative behaviors (see Chapter 3).
Figure 6.2. Chromatin immunoprecipitation. In the oxytocin receptor promoter (A) (Oxtr prom), the magnitude of the estrogen effect depends on which promoter segment is studied, which histone modification is analyzed, the neuronal group studied (B) VMH, ventromedial hypothalamus, VMH. (cf. C) Preoptic area (POA) and the time in hours after estrogen (E2) treatment. Histone marks: Histone H3 acetylation (H3Acetyl); histone 3 lysine 4 trimethylation (H3K4me3). (Adapted from Gagnidze et al. 2013.)
In view of the data that OTR can signal through protein kinase C (PKC) isoforms, we looked at coexpression of selected PKC isoforms in the same individual neurons. The most discriminating analysis was for triple coexpression of ERs, OTR, and each selected PKC isoform. These patterns of triple coexpression were significantly different for male versus female VMH neurons. Further, individual neurons expressing ER-α could distribute their signaling across the various PKC isoforms differently in different cells, whereas the reverse was not true. On a VMH nerve cell-by-cell approach, therefore, oxytocin signaling plays a prominent role.
The OTR system in VMH could be studied electrophysiologically as well (Kow et al. 1991). The positive effect of oxytocin on lordosis is dependent on priming by ovarian steroids, estrogens, and progesterone. These steroids modulate oxytocin binding in specific brain nuclei, including the ventrolateral portion of the ventromedial hypothalamic nucleus (vlVMH). So we used microelectrodes to record neurons in the vlVMH in hypothalamic slices to characterize the electrophysiological actions of oxytocin. To examine the effects of ovarian steroids on oxytocin actions, we used brain slices prepared from ovariectomized rats either treated with estrogen or not, and some slices were treated with progesterone in vitro. Oxytocin affected the electrical activity of large numbers of vlVMH units. Of those neurons affected, 94 percent responded with excitation. This predominant stimulatory action of oxytocin was consistent with its lordosis-facilitating effect because increases in the activity of VMH neurons are associated with the facilitation of lordosis. Pharmacological analyses with selective oxytocin agonists and antagonists as well as structurally related peptides showed that the excitatory action of oxytocin is mediated by OTR.
In these electrophysiological studies, it was gratifying that estradiol modulated several aspects of oxytocin transmission. First, it increased neuronal responsiveness to oxytocin, especially at the lowest concentration used (0.2 nM). In addition, it caused neuronal responses to oxytocin to correlate significantly with responses to acetylcholine and norepinephrine, which also can act on the ventromedial hypothalamus to facilitate lordosis. Finally, estradiol enhanced the excitability of laterally projecting neurons, which have been implicated in lordosis. In estrogen-pretreated slices, the addition of progesterone in vitro caused little further effect on the responses of individual neurons to exogenous oxytocin. Altogether, these hard-won electrophysiological findings have been consistent with the hypothesis that estrogen potentiates oxytocin action by increasing functional OTR preferentially in lordosis-relevant neurons, thereby enabling oxytocin to efficiently facilitate female reproductive behavior.
Further, as documented in Chapter 3, we used in situ hybridization to study oxytocin gene expression in preoptic and hypothalamic neurons as well as to determine estrogenic influences on oxytocin expression. We found that estrogens caused a 75 percent increase in the number of grains per cell (i.e., oxytocin mRNA levels per cell). Likewise, estrogens caused a 4.4 times increase in OTR mRNA levels in the VMH.
Even as oxytocin administration to the hypothalamus had been shown strongly to facilitate lordosis behavior, we wanted to try the opposite approach—to reduce lordosis behavior. Thus, we used infusions of antisense oligodeoxynucleotides directed against OTR mRNA, microinjected them directly into the VMH, and blocked lordosis behavior (McCarthy et al. 1994).
Thus, as I summarized in Chapter 3, knowing that (a) estrogens increase transcription rates in both oxytocin and OTR systems, and (b) oxytocin working through its OTR facilitates lordosis, it follows that (c) one way in which estrogens increase lordosis behavior is by revving up, by increasing the rate of transcription of oxytocin and the OTR genes. Further, positive estrogenic effects on oxytocin as well as OTR transcription could have multiplicative effects.
Expansion to Other Social Behaviors
Robert Bridges at Tufts University, Michael Numan at Boston College, and Curt Pedersen at the University of North Carolina pioneered studies of maternal behaviors in laboratory animals. Rockefeller graduate student Susan Fahrbach beca
me interested when she showed that very low amounts of estradiol could be implanted among neurons in the preoptic area—neurons that Numan had shown to be critical for maternal behavior—to significantly facilitate maternal behavior in female rats (Fahrbach and Pfaff 1986). She used females that had had no pregnancies and no experience with pups and thus would be unresponsive. But given these minute preoptic area implants, they responded significantly more quickly. She also showed short-latency maternal behavior in such inexperienced females if and only if the females were primed with estradiol (Fahrbach, Morrell, and Pfaff 1984). Then she combined estrogen receptor autoradiography with retrograde neuroanatomical methodology to identify (Fahrbach, Morrell, and Pfaff 1986b) those estrogen-binding pro-optic neurons that project axons to the midbrain through a route (discovered by Michael Numan during his sabbatical with us) to be crucial for maternal behavior (Numan, Morrell, and Pfaff 1985).
Susan then exploited the opposite approach: the use of OTR antagonists to reduce maternal behavior (Fahrbach, Morrell, and Pfaff 1985a,b). Using the cerebral ventricles as an optimal route, she applied, in separate experiments, antisera to oxytocin and an OTR antagonist and measured maternal behavior in an animal preparation well known reliably to yield high level, short latency maternal behavior. The OTR antagonist multiplied the latency to get maternal behavior by approximately four times, and the antisera to oxytocin doubled the latency.
Ana Ribeiro and I followed up Susan’s finding by using an adeno-associated viral vector encoding an antisense sequence directed against ER-α and microinjecting it specifically into the medial preoptic area (Ribeiro et al. 2012). In doing so we abolished all measures of maternal behavior: females did not retrieve pups, they did not lick or groom pups, and they did not assume nursing postures. Thus, the estrogen / oxytocin theme—the essentiality of the estradiol / oxytocin combination—remains as strong for maternal behavior as it does for mating behavior.