Acknowledgments
We would like to thank Dr. Zhongmin Lu and Seth Tomchik for use of histological equipment, confocal and light microscopes and especially for technical and intellectual support.  We thank Dr. Jim OÕReilly and the OÕLab for support.  A special thanks to Alexandra Sterlin for support and delivery of literature.  A hearty thanks to G-Rob Burgess for technical support. MM and LG would also like to extend thanks to the University of Miami Department of Biology for travel support as well as the UM College of Arts and Sciences Kriloff foundation for travel funding.  LG is especially grateful to SICB for student support for this meeting.  This study was supported by the EPA STAR-GRO fellowship number 91621401 for LG.

The brain illustration for this poster was drawn by Mark Mandica.  Please visit www. mandica.com for further information.  You can contact MM at mandica@bio.miami.edu or LG at ganser@bio.miami.edu.  Visit us at the O'Lab website www.bio.miami.edu/oreilly or www.bio.miami.edu/zlu.
Abstract
Adult cane toads (Bufo marinus) display sexually different reproductive behaviors.  Male toads call females to mate.  Female toads respond by moving toward the male`s call, a response known as call phonotaxis.  Sexual differences exist in the areas of the brain that govern these behaviors.  In this preliminary study we have identified the sexually different areas of the brain of Bufo marinus.  We have used histological techniques to describe the general neuroanatomy of the cane toad then pinpoint areas that differ in size, morphology, and cell body size and population.  During development, genetic and sex hormonal cues contribute to the formation of these male or female-like brain areas.  Thus we hypothesize that areas of the brain that are affected by sex hormones may also be affected by endocrine disrupting chemicals in the environment.  These preliminary data will help us to recognize any effects of the controversial endocrine disrupting herbicide atrazine on the sexual differentiation of the brain of the Bufo marinus.
Introduction
Previous studies by Boyd et al. (1992) and Gonzales and Smeets (1992) described the neuroanatomical locations of arginine vasotocin immunoreactive (AVT-ir) cells and fibers.    From these studies we were able to identify nuclei in the brain of Bufo marinus.
The endocrine system serves as one of the main control systems of the body.  During the amphibian larval period, hormones help to organize bipotential reproductive tissues in the brain and the gonads into their male or female forms.  It is during this critical period of organization that certain man-made chemicals may disrupt the normal endocrine cascade that governs the function and form of reproductive tissues.  These endocrine disruptors, often found in agricultural chemicals and industrial wastes, have been known to affect the morphology of the gonads as well as the mating behaviors of exposed animals. 
     The putative endocrine disruptor, atrazine, is liberally used on crops and residential grasses in South Florida.  It is the most commonly used herbicide in the United States and is specifically used on South FloridaÕs sugar cane crop (Gross et al. 2003), on recreational grasses and residential lawns.  Cane toads (Bufo marinus) are an invasive species in Florida and flourish in developed and agricultural areas where herbicide use is common.  Cane toads collected from agricultural areas in South Florida, showed evidence of abnormal gonad morphology, while toads collected from reference areas appeared normal (McCoy et al. 2002).  A previous study (Hayes et al. 2002) concluded that atrazine feminized the gonads of male clawed frogs (Xenopus).  
Male bufonids possess vestigial ovarian tissue (BidderÕs organ) that may increase susceptibility to disruption by endocrine disruptors.  We hypothesize that atrazine will affect the morphology of the gonads as well as the areas of the brain that govern mating behavior.  In this preliminary study, however, we compare neuroanatomical structures of male and female Bufo marinus.  We use Nissl stain to identify common nuclei and to make qualitative comparisons of nucleus size, cell body size, and cell density between males and females. 

Methods
Cane toads were collected from various sites in South Florida and euthanized for brain and gonad analysis.  General neuroanatomical observations were made on tissues stained with Cresyl Violet stain for Nissl substance.  Brains were dissected from cane toads, fixed in 4% paraformaldehyde, and embedded in gelatin with 30% sucrose.  Tissues were post-fixed in 30% sucrose with 4% paraformaldehyde.  Serial frozen sections (50 μm) were made on a sliding microtome.  Cresyl violet-stained tissues were viewed with a Nikon Eclipse E600 light microscope and analyzed using Neurolucida software.  Qualitative comparisons between males and females using differences in nucleus size, soma size, and cell body density were made on Nissl stained tissues.  We identified areas of the brain potentially responsible for mating behavior using immunohisto-chemistry for arginine vasotocin (AVT). These data for AVT are not included in our results.
Conclusions
Previous studies by Boyd et al. (1992) found sex differences in AVT-ir cell body and fiber density in the amygdala pars lateralis and habenula regions of the Rana catesbeiana brain.  We were unable to notice any qualitative sex differences in cell body size, cell body density, or nucleus size in these areas of male and female Bufo marinus brains.  We did, however, note some subtle differences in cell body density and cell body size in the glossopharyngeal nerve area of the male brain.  Cell bodies in the solitary tract seem more organized in the male brain compared to the female brain. 
Our preliminary studies using Cresyl Violet stain for Nissl substance have helped us to identify neuroanatomical structures and subtle sex differences in the cane toad brain.  It is clear that meticulous study of sexually different areas of the anuran brain that have been previously identified are necessary.  Our initial attempts at immunocytochemistry for AVT showed immunoreactive labeling of various cell bodies and fibers throughout the brain.  However, we must be able to pinpoint specific nuclei that are AVT immunoreactive.  Future immuno-cytochemical studies for AVT paired with the identification of nuclei in the cane toad brain will help us to identify sex differences in areas of the brain that govern mating behavior. 
Our future studies will include repeated immunocytochemical studies for AVT-ir cell bodies and fibers in the brain of Bufo marinus, making note of any sex differences in cell body and fiber size and density.  These studies will aid in our study of the effects of the putative endocrine disruptor, atrazine, on areas of the brain that mediate mating behavior.

Literature cited
Boyd, SK, CJ Tyler, GJ De Vries.  1992.  Sexual dimorphism in the vasotocin system of the bullfrog (Rana catesbeiana).  J. Comp. Neurol.  325:  313-325.
Gonzales, A and WJAJ Smeets. 1992.  Comparative analysis of vasotocinergic and mesotocinergic cells and fibers in the brain of two amphibians, the anuran Rana ridibunda and the urodele Pleurodeles waltlii.  J. Comp. Neurol.  315:53-73.
Gross, TS, KA McCoy, M Sepulveda, JA Carr, JP Giesy, AJ Hosmer, RJ Kendall, K Solomon, EE Smith, and G van der Kraak.  2003.  Atrazine exposure and the occurrence of reproductive abnormalities in field caught Bufo marinus from South Florida.
McCoy, KA, M Sepulveda and TS Gross. 2002. Atrazine exposure and the occurrence of reproductive abnormalities in field caught Bufo marinus from South Florida. In: Proceedings of the Society of environmental Toxicology and Chemistry, 23rd Annual Meeting.

Assessing Sex Differences in the Brain of the Cane Toad, Bufo marinus
Lisa R. Ganser and Mark L. Mandica.  Department of Biology.  University of Miami.  Coral Gables, FL 33124

Results