S Marc Breedlove - US grants

The striated bulbocavernosus (BC) muscles of the rodent perineum are innervated by motoneurons in the spinal nucleus of the bulbocavernosus (SNB). In adulthood, the BC muscles are present in males only. However, newborn female rats have BC muscles and SNB cells have made both anatomical and functional contact with them. Nevertheless, both SNB motoneurons and BC muscles will degenerate in females unless androgens are administered perinatally. There is evidence that such androgen treatment acts primarily on the BC muscles themselves, since the muscles are spared by androgen after the loss of supraspinal neural afferents or even the entire lumbosacral spinal cord. Two experiments are proposed to confirm or refute the target muscles as the primary site of androgen action upon the SNB system. One experiment will attempt to spare montoneurons which are themselves genetically incapable of responding to androgen. The second experiment will attempt to masculinize the SNB system by providing androgen responsive BC muscles transplants to androgen-insensitive newborn males. The possiblity that BC muscles produce proteins which are retrogradely transported by developing SNB cells will be investigated by amino acid autoradiography. Perinatal treatment with the steroid DHTP can alter the final spinal location of SNB cells as determined by previous retrograde tracing studies. There is suggestive evidence that DHTP accomplishes this abnormal spinal location by affecting the migration of motoneurons. Two experiments will test this hypothesis. Finally, the sexually dimorphic character of motoneuronal groups innervating perineal muscles seems to be common in mammals, since the homologue to the SNB, Onuf's nucleus, has more cells in males than in females in both dogs and humans. Furthermore, the number of Onuf's motoneurons is altered by perinatal androgen in dogs. We will conduct androgen autoradiography to determine whether motoneurons in Onuf's nucleus accumulate androgen.

DESCRIPTION (provided by applicant): We will examine the influence of steroid hormones, specifically androgens, on a neuromuscular system that responds to these hormones throughout life. Previous work has shown that androgens maintain the bulbocavernosus (BC) and levator ani (LA) muscles of developing male rats. These striated perineal muscles are normally present in females at birth but, in the absence of androgen, the muscles die. A single injection of androgen to newborn female rats will preserve the muscles for life. The BC/LA muscles are innervated by motoneurons in the spinal nucleus of the bulbocavernosus (SNB). Androgenic sparing of the BC/LA target muscles also indirectly spares the SNB motoneurons from developmental apoptosis. The present project will study the effects that androgenic hormones continue to exert on the SNB system in adulthood. Castration of adult male rats triggers a host of changes in the structure and function of the SNB system. For example, the somata and dendrites of SNB motoneurons, as well as the neuromuscular junctions (NMJs) these motoneurons make upon the target muscles, shrink following castration. Testosterone (T) therapy can prevent and/or reverse this shrinkage. We have found that T cannot maintain SNB somata size when we pharmacologically block the N-methyl-d-aspartate (NMDA) type glutamate receptor. This effect of NMDA blockade seems to be specific to SNB motoneurons and to the effect of T. We propose to determine whether NMDA blockade also prevents the effects of T on SNB dendrites and NMJs. We have found that SNB motoneurons, but not other nearby motoneurons, increase their expression of the gene for the NMDA receptor subtype 1 (NMDAR1). So the androgen-induced increase in NMDA receptor stimulation of SNB motoneurons may contribute to maintaining soma size. We will use a mosaic analysis to ask whether androgen acts directly upon SNB motoneurons to increase their expression of the NMDAR1 message. We will also determine whether SNB motoneurons express a newly discovered NMDA receptor subtype (NMDAR3), whether androgen manipulations alter NMDAR3 expression, and if so whether this is a cell-autonomous response of the motoneurons. These studies should lead to a better understanding of neuromuscular systems, of the influence of steroid hormones on the spinal cord, and the etiology of several neuromuscular disorders including amyotrophic lateral sclerosis and Kennedy's syndrome.

We will study two brain regions that are structurally different in male and female rats. These examples of sexual dimorphism offers a means of studying the relationship between neuroanatomical measures and behavior. Any sexual dimorphism that is found in the brain could potentially contribute to sex differences in behavior. We will focus upon the hippocampal formation, which has been implicated in spatial cognition in mice, rats and humans. Spatial cognitive abilities have also been found to differ between the two sexes in all three species. We have recently replicated previous reports of sexual dimorphism of this structure in all three species. We have recently replicated previous reports of sexual dimorphism of this structure in rats and will apply our expertise in behavioral endocrinology to determine when the sexual dimorphism arises, whether hormones play an important role in the masculinization of the structure and, if so, which hormone receptor is activated in vivo. We will also examine the medial amygdala, which has been implicated in both masculine copulatory behavior and juvenile play behavior in rats. We recently replicated the reported sexual dimorphism in the overall volume of this nucleus but were surprised to find that it was feminine in rats with a defective androgen receptor. As with the hippocampus, we will examine when and how the medial amygdala comes to develop differently in male and female rodents. Such analyses may offer insights into possible mechanisms underlying sex differences in human behavior and psychopathology.

There continues to be a shortage of young people, particularly young people from under-represented groups, entering careers in basic research. One strategy to boost these numbers is to provide programs that discuss science generally. However, such programs suffer by being so broad in subject matter that they might not engage students' interest in scientific problems, and being so vague in terms of what it's like to be a scientist that the students have only a theoretical notion of what such a career entails. An alternative strategy is to identify a particular field of study, find students who have already shown some interest in that particular field, and then bring them together to give them hands-on experience in a lab and ample social opportunities to meet and talk to active scientists in that field. In partnership with a consortium of universities, including several traditionally African-American undergraduate colleges, we will bring 10 undergraduate students from around the country to our campus for a one-week intensive course in behavioral neuroendocrinology: the study of the influence of hormones on behavior, and the influence of behavior on hormone secretion. We will identify the students by soliciting from laboratories engaged in behavioral endocrinology research. In our inaugural session offered last year, we received over 50 strong candidates for 8 positions. In order to capitalize on this learning experience, and to give the students a deeper understanding of the field, as well as a sense of what life as a scientist is like, we will send these students to the subsequent meeting of the Society for Behavioral Neuroendocrinology (SBN). At the SBN meeting, the students have the opportunity to have "lunch with the professors" from yet other universities and discuss current and future scientific endeavers. We are requesting funds to pay the airfares, registration and lodging for these undergraduates for both the short course on campus and the SBN meeting thereafter, as well as funds to bring neuroendocrinologists from other campuses to serve as instructors. What is unique about this experience is the hands-on laboratory training, the intensive social interaction with scientists, from half a dozen different universities, before and during a professional meeting, and the exposure to a wide range of working graduate students and postdoctoral fellows in the field. Bringing together an mixture of students, emphasizing students from under-represented groups, who have expressed interest in a career in science, will increase the strength and the diversity of the applicant pool in this area of science nationwide. Our goal is to let these students see how intellectually and socially satisfying a career in science can be so that more of them choose this career.

? DESCRIPTION (provided by applicant): Among the most prevalent of mental afflictions, anxiety disorders are more commonly diagnosed in women than in men. There is ample evidence that circulating levels of gonadal steroid hormones, including estrogens such as estradiol and androgens such as testosterone, can reduce anxiety in humans and so may play a role in the sex difference in anxiety disorders. Laboratory mice also show a sex difference in anxiety- like behaviors that, as with anxiety in humans, are reduced by circulating testosterone. In mice, it is clear that circulating levels of steroids modulate anxiety-like behaviors. We want t perform exploratory experiments to determine which brain regions are being affected by hormones to reduce anxiety-like behaviors in mice. Preliminary observations suggest that two plausible brain sites for modulating anxiety, the medial prefrontal cortex and the hippocampus, are not the sites responding to testosterone. Other observations implicate the basolateral amygdala as a potential site of hormone action and we would like to conduct experiments to confirm or refute these indications. Understanding how gonadal hormones act on the brain to reduce anxiety in mice may suggest new therapeutic approaches for anxiety disorder in humans.