Gerald Brian Downes - US grants

Gerald B. Downes, Proposal Number 0527922
Analysis of Glycinergic Neurotransmission in the Early Zebrafish Spinal Cord

Vertebrate locomotive behavior, such as walking or swimming, consists of repetitive and alternating muscle contractions that are controlled by groups of neurons within the spinal cord. These neurons, collectively known as the spinal cord central pattern generator (CPG), are essential for locomotive behavior. Early in animal development the emergent spinal cord CPG is also thought to orchestrate the repetitive spontaneous movements of the limbs or tail, that are the first behavior observed in all vertebrate embryos. In both the early and more mature forms of the spinal cord CPG, the neurotransmitter glycine plays crucial roles in regulating and coordinating neuronal activity and these roles likely change during the course of development. Although glycine neurotransmission is present in the early spinal cord CPG, key features about the role of glycine remain unknown. The function of glycine within the early spinal cord CPG is not known, not all of the genes and cells required for glycine function within the early spinal cord CPG have been identified, and it is not understood how the role of glycine changes during development. The zebrafish embryo provides several advantageous features to analyze spinal cord CPG development and address these issues. In this proposal, a combination of molecular, genetic, and physiological approaches unique to zebrafish will be employed to elucidate the genes and cells required for glycine function during spontaneous movement of the tail of the embryo, the first behavior observed in zebrafish development. The results from this study will identify essential components of the early spinal cord CPG, and will provide a foundation for future work to examine how this network is modified during development to yield the mature spinal cord CPG.
Since spinal cord organization is thought to be broadly conserved among vertebrates, this work holds promise to provide insight into spinal cord development and function in higher vertebrates. In addition to satisfying research goals, these studies will also greatly aid in satisfying teaching goals. Both graduate and undergraduate students will receive hands-on, laboratory training by contributing to this project.

DESCRIPTION (provided by applicant): Vertebrate locomotive behavior, such as swimming or walking, requires the precise formation of neural networks within the spinal cord. Although the broad organization of these spinal cord networks is understood, little is known about how these networks are first established and then modified during embryogenesis to produce a mature spinal cord network. This proposal requests a mentored "Career Development Award to Promote Diversity in Neuroscience Research" (K01) to investigate spinal cord network formation in the zebrafish embryo. The applicant is Gerald B. Downes, Ph.D., a recently appointed Assistant Professor in the Biology Department at the University of Massachusetts Amherst. This award will enable Dr. Downes to acquire expertise in microscopic imaging of spinal cord neuron activity in vivo. This powerful technique will complement his existing expertise in genetics and molecular biology, and will allow for a multifaceted approach to examine spinal cord development. Thomas Zoeller, Ph.D. and Peter Hepler, Ph.D., a well-established developmental neurobiologist and live-cell imaging physiologist respectively, will serve as the mentor and co-mentor. Combined, they have substantial experience in developmental neurobiology, imaging cellular activity and mentoring, and will greatly facilitate Dr. Downes' long-term career goal to establish a competitively funded research program. The central research goal for this proposal is to identify genes and cellular events required for the development of spinal cord networks. The experiments in this proposal utilize two members of the accordion class of zebrafish mutants, bajan and squeezebox. These mutants demonstrate aberrant locomotive behavior, consistent with them harboring spinal cord network defects. Neither the mutated gene nor the cellular defect is known in either mutant; therefore they can be effective tools to elucidate the potentially novel genes and cellular events essential for spinal cord network development. Since spinal cord organization is broadly conserved among vertebrates, these studies may provide insight into human spinal cord development. The experiments in this proposal will: 1) test the hypothesis that the bajan and squeezebox genes contribute to spinal cord network formation using imaging approaches; 2) resolve the development of abnormal locomotive behavior in each mutant; and 3) determine the molecular identity of both the bajan and squeezebox genes.

Walking and swimming are produced by the coordinated activity of brain cells called neurons that communicate with each other using chemical signals known as neurotransmitters. The chemical signals are passed from one neuron to another across a network of neurons in the brain, then on to neurons in the spinal cord, and finally, the signal is passed to muscles. Some neurotransmitters increase neuron activity, whereas others decrease neuron activity. It is not known how different neurotransmitters balance their effects to coordinate neuron network activity and enable normal locomotion. The goal of this project is to investigate the role of one particular neurotransmitter in the brain to better understand how it regulates locomotion. The project will be carried out using zebrafish because this vertebrate animal has a more simple brain and spinal cord organization than mammals, and it is transparent, which makes it easy to examine under a microscope. Due to the many similarities in neurotransmitters and locomotor networks between zebrafish and mammals, this work can have wide-reaching implications. This project is a collaboration between four faculty researchers from three colleges. Undergraduates at each institution will participate in the research and have access to the expertise across institutions. The research team, including faculty, graduate and undergraduate students, will perform teaching demonstrations about neuroscience at nearby middle and high schools, and students from these schools will visit the investigators' laboratories. Through this outreach effort, 300-600 primarily underrepresented minority middle and high school students will be exposed to scientists and neuroscience.

The central hypothesis that will be tested in this project is that, during early larval stages, a limited number of gamma amino butyric acid (GABA) type A receptor isoforms regulate locomotor networks through a small number of hindbrain reticulospinal neurons. To test this hypothesis, the expression of GABA-A receptor subunits will be determined, the effects of genetic inactivation of GABA-A receptor subunits on locomotor network activity and behavior will be investigated, and the contribution of select hindbrain neurons in generating GABA-A receptor-mediated hyperactive behavior will be assessed. These studies will be carried out in genetically modified zebrafish using microscopy, electrophysiological recordings, quantitative analysis of locomotor behavior, and a novel photochemical approach that enables control of specific GABA-A receptors using different wavelengths of light.