The Journal of Undergraduate Neuroscience Education (JUNE), Fall 2003, 2(1): A28-A35 JUNE is a publication of Faculty for Undergraduate Neuroscience (FUN) www.funjournal.org Sex Differences and Organizational Effects of Androgen in Spinal Cord Motor Nuclei William Grisham, Heidi B. Jones, & Sun Hee Park Department of Psychology, University of California, Los Angeles, Los Angeles, CA 90095-1563 This article describes a laboratory module taught at UCLA and offers digitized microscope images that will allow instructors to recreate this module at their home institutions with only a computer required. This module allows for 1) an exploration of the effects of hormones on neural development, 2) the demonstration of sex differences in the nervous system, 3) the production of robust and statistically significant data by novice undergraduates, 4) the discussion of sophisticated statistical analyses (ANOVAs with significant main effects and an interaction), and 5) the understanding of at least some of the neuroanatomy of the spinal cord. Specifically, this module both replicates and extends a previously published experiment on sexually dimorphic neurons in the spinal cord of rats (Grisham et al., 1992), which examined the effect of antiandrogen exposure (Flutamide) in utero on sexually dimorphic spinal motoneurons in male and female rats. Key words: CNS development, sex differences, hormones, spinal nucleus bulbocavernosus, spinal cord, organizational effects Undergraduate neuroscience laboratories rarely tackle developmental questions in mammals that require time for the animals to mature. Many undergraduate neuroscience laboratories do not have necessary resources to establish breeding colonies, perform histological preparations, and do analyses at a microscopic level. Also, finding laboratory experiences that naïve students can perform and obtain meaningful, statistically significant data are challenging. In order to address these needs, this article describes a module taught at UCLA that invariably produces statistically significant, meaningful data. This module involves blocking androgen receptors in development and examines the consequences on spinal motor neurons. The need for a breeding colony, histological preparation, and microscopes has been removed because this article and its supplement offer a set of digitized microscope images that will enable instructors to recreate the module at their home institution with only a computer (see Appendix A). Steroid hormones, particularly testosterone and its metabolites (i.e. estradiol and dihydrotestosterone), play two different roles in development of the nervous system: organizational roles and activational roles (cf. Phoenix et al., 1959; Arnold & Gorski, 1984; Nelson, 1999). The organizing actions of steroid hormones happen early in life, during a critical/sensitive developmental period, and are relatively permanent. Activational actions happen later in life, are less closely tied to a given developmental period, and the effects are relatively transient, following the levels of steroids present (c.f. Nelson, 1999, chapter 3, for a review). This laboratory is designed to illustrate the organizational effects of gonadal steroids and the consequent sex differences that they can produce in the nervous system. Specifically, this module replicates the findings of a sex difference in neurons of the spinal nucleus bulbocavernosus (SNB) and the demasculinizing effect of Flutamide, an antiandrogen (Grisham et al., 1992). This module extends previous findings by looking for sex differences and the effects of antiandrogen exposure on a second motoneuron pool, the retrodorsal lateral nucleus (RDLN). SNB neurons are large, densely-staining neurons found in the lumbar spinal cord within about 200 microns ventral to the central canal (Figure 1). These motor neurons innervate the muscles of the penis, the bulbocavernosus (BC), and the levator ani (LA) (cf. Breedlove, 1984, for a review), which are sexually dimorphic: males have them but they are absent or vestigial in adult females. SNB neurons, not surprisingly, are larger and more numerous in males than they are in females (Breedlove & Arnold, 1980; Grisham et al., 1992). While this may seem like a trivial finding, the long-held belief was that there were no anatomical sex differences in the nervous system, only physiological sex differences (cf. Phoenix et al., 1959). SNB neurons are under hormonal control in the course of development. Administration of testosterone in development masculinizes the female SNB (Breedlove, et al., 1982). Although this result suggests that androgens during development are responsible for the sex difference observed in adulthood, it does not necessarily prove that differential androgen circulation during development is necessary for the sex difference to occur. Documented sex differences in androgen levels occur prenatally and may be the source of sex differences in the developing central nervous system (Weisz, & Ward, 1980). The ideal experiment would eliminate the source of androgens by castrating the males in utero and examine the result. Since this surgery is a difficult if not impossible procedure, in place of castration, the actions of androgens can be antagonized by Flutamide, a drug that blocks androgen receptors by preventing the binding of androgens. This module contrasts SNB neurons that innervate a highly sexually dimorphic BC/LA muscle with RDLN neurons, motor neurons that innervate the flexor digitorm brevis (FDB) muscle (Leslie et al., 1991). Although the FDB muscle is slightly different between the sexes (Leslie