This project studies the changes that occur in the structure of the brain when an adult animal learns to do something new. Young honey bees work within the hive, but at approximately three weeks of age begin to forage to flowers for pollen and nectar. Changes in the size of individual nerve cells and the volume of entire brain regions result from the shift from in-hive tasks to foraging. This project investigates the role of hormones in promoting brain growth in response to new experiences using the honey bee model. The responses to important developmental hormones of bee neurons growing in culture will be studied using both microscopy and methods that permit gene expression to be measured. These studies will yield an understanding, at a cellular and molecular level, of why nerve cells are more likely to grow during some stages of life than others. This project will provide training in neuroscience research for a graduate student. In addition, the co-PIs will develop a new bioinformatics course for undergraduates. Teaching materials developed for this course will be made freely accessible to all interested teachers and students via a bilingual (English/Spanish) web site maintained at Wake Forest University.
The structure and function of all animal brains change in response both to developmental signals, such as hormones, and experience (what the animal actually does). The mushroom bodies are a region of the honey bee brain that serve as a model for investigations of neuronal plasticity from the behavioral to the molecular levels of analysis. In honey bee colonies, a critical feature of social organization is age-based division of labor among worker bees. Workers tend the queen, rear larval brood, and maintain the physical structure of the hive for the first 2 to 3 weeks of adult life, then switch to foraging outside of the hive for their final 1 to 3 weeks. Studies of regional brain volumes have revealed that foragers have predictable changes in brain structure relative to non-foragers, including growth of the mushroom bodies, which are essential for learning and memory. This growth has been shown to reflect growth of the dendrites of the mushroom body neurons. In addition to this experience-dependent dendritic growth, there is also an early (first week of adult life) experience-independent phase of dendritic growth. This growth occurs against a well-characterized endocrine backdrop. Small peaks of the steroid ecdysteroid hormone are secreted during the first 3 days of adult life, followed by a relatively hormone-free period while the bee works within the hive. Foragers typically have undetectable levels of ecdysteroids. Ecdysteroids signal by binding to members of the nuclear receptor superfamily of proteins, in an action similar to the steroid hormone receptors of vertebrates. The focus of this project was to identify the mechanisms by which mushroom body neurons in the adult honey bee brain respond to the ecdysteroid hormones. The major questions asked were how ecdysteroids regulate gene expression in the adult brain and whether the effects of ecdysteroids are different at different stages of development. To study the effects of the hormone at the level of gene expression, a system of primary neuron culture was developed that permitted measurement of growth and extraction of RNA in isolated neurons. In addition, past research had shown that multiple forms (isoforms) of honey bee ecdysteroid receptors are encoded in the honey bee genome, and an additional goal was to ask if one isoform of the receptor was more important than the others in the regulation of dendritic growth. It was also important to develop reliable techniques for measurement of ecdysteroids in small blood and tissue samples taken from individual bee. These data provide important context for the results obtained from the neuron cultures. The technical challenges of this project were met (development of new techniques for culturing insect neurons, adaptation of an ecdysteroid assay for use with honey bee tissues). This led to the following findings: signaling via the ecdysteroid receptor is required for outgrowth of neuronal processes; one of the genes regulated by ecdysteroids is a cytoskeletal protein, beta-tubulin; and a specific receptor isoform (EcR-A) is expressed most abundantly in the mushroom body neurons at the time when plasticity is maximal but ecdysteroid levels are low. This unexpected result suggests a possible function for the unliganded (no hormone bound) receptor in the regulation of neuronal growth. The method for primary neuron culture developed for this project has already been used for other purposes, such as screening for the direct effects of insecticides on the brains of beneficial insects. The broader impacts of this project were the development of a bioinformatics for beginners seminar course for college freshmen based on studies of honey bee gene expression and behavior, the training of a graduate student, and the participation of undergraduates in the research aspects of the project. In addition, numerous presentations on the science of beekeeping were made to county beekeeping associations and at meetings of the North Carolina Honey Bee Research Consortium, and the laboratory hosted Campus Day visits for high school students to raise awareness of insect molecular biology.
