Ginger S. Withers - US grants

Nerve cells (neurons) in the mammalian brain often have elaborate treelike branching of fine cellular extensions called dendritic arbors, which receive the chemical signals of activity from other neurons. Like an antenna, the location and number of branches on a dendritic arbor organize the pattern of the afferent input, so the decision about where and when to form a branch is critical. The intracellular structures of these dendrites include cytoskeletal components called actin and microtubules, and these components are dynamic, changing the dendritic structure over time by elongation or retraction or stabilization of the branches at particular points. It remains unclear what the mechanisms are that determine the patterning of these often highly specific arbors during development and growth. To approach this question, this project combines several recent technical advances that give the ability to 1) accelerate dendritic development significantly in cultured neurons from the hippocampal region of the brain; 2) follow the expression and trafficking of proteins and organelles within the living neuron by high-resolution microscopic time-lapse imaging; and 3) to create patterned substrates, using novel creative nanofabrication techniques to provide external cues for cellular development. The experiments will determine the roles of actin and microtubule dynamics, and the influence of external cues such as cell adhesion molecules and location of afferent contacts from other neurons, on dendritic branching.
Results will provide a major advance on an important topic of neural development where fundamental information is still unknown, and the interdisciplinary approach will enhance interactions between biology and engineering. The excellent integration of educational activities with the research will have a major impact on research experience in this liberal arts college, and even on the local public schools.

DESCRIPTION (provided by applicant): Dendrites form the primary receptive surface for synaptic connections, but key questions still remain about how dendrite field size, geometry and synaptic density are regulated. In the context of development, data obtained from addressing these questions would be immediately relevant to a wide array of human neurodevelopmental disorders. Further, mechanisms of initial morphogenesis of postsynaptic structure are thought to have mechanistic parallels between later experience-dependent plasticity associated with memory and recovery from brain injury. Here, we focus on the role of the astroglia cell, a newly-recognized partner in synapse formation, in regulating neuron development. Proposed experiments exploit primary cultures of dissociated hippocampal neurons as a bioassay to detect the influence of astrocytes on synaptic formation between developing neurons. Our preliminary data confirm previous reports from retinal ganglion cell culture that soluble factors released by astroglia regulate synapse formation. We also have evidence that astroglial secretions inhibit dendritic growth in a manner that may shift neurons from an intrinsic program of branch formation to an extrinsically-mediated readiness for synapses. In Specific Aim 1, we will manipulate neuron-glia interactions, comparing conditions that model the in vivo state of physical contact to a co-culture condition that allows only soluble signals from glia to reach neurons. Aim 2 uses markers of pre- and postsynaptic specializations to determine if astroglia differentially affect maturation of these compartments in excitatory vs. inhibitory neurons. Guided by the data of Aims 1 and 2, Specific Aim 3 will test individual glial signals with over-expression and RNAi knock-down experiments. PUBLIC HEALTH RELEVANCE: The proposed studies characterize the relationship between glial cells and synapse development. There is a diverse array of postnatal neurodevelopmental disorders (mental retardation, Rhett's syndrome, autism) as well as many genetically-determined disorders (e.g. Down's and Fragile X) that are poorly understood at the cell biological level, but clearly associated with abnormal synapse formation. Previous studies have focused exclusively on neurons, but the active role of glial in regulating neuron maturation can no longer be denied. The proposed work will advance our understanding of the mechanisms by which glial cells influence synapse formation, to further our understanding of normal and pathological neural development.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

This award funds the acquisition of a new scanning electron microscope (SEM) at Whitman College. Whitman College has a track record of 24 years of successful research and student training using scanning electron microscopy. The college's previous SEM is no longer functional, and new faculty research programs require the acquisition of a new instrument capable of capturing higher resolution images and handling wet biological samples, such as plant embryos and cultured neurons. Features of this new SEM will allow diverse applications: very low vacuum will allow research of hydrous Arabidopsis samples for studies of gene functions in plant development, and the three-nanometer resolution will enable high-quality digital photos of brain cells, evolutionary adaptations of snake fangs, surfaces of protein crystals, and traces related to the function and manufacture of prehistoric tools; these are all components of current, student-centered research programs. Furthermore the award funds an energy dispersive spectrometer (EDS) attachment that will acquire and map chemical compositions of stone tools for provenance studies, compositions of minerals in volcanic rocks for estimates of pressure and temperatures of pre-eruption crystallization, and compositional variation of carbonate-rich root casts for carbon cycle studies. This enhanced SEM therefore will strengthen the research programs of eight faculty members, including two junior faculty, in three different departments (Biology, Geology, Anthropology), and it will contribute to cross-disciplinary research. Research results will be presented at national and regional meetings, submitted for publication in disciplinary journals, and featured on the Whitman College website.

The SEM will also have considerable broader impacts. Whitman College has an excellent record of training student researchers, with 50% of students participating in research with the investigators going on to graduate or professional schools. More than 50% of students involved in research have been women or members of underrepresented groups. Because SEM is used in a wide variety of private, governmental, and academic research fields, use of this instrument in multiple upper-level courses, as well as research, will prepare students for sophisticated post-graduate work. Acquisition of an enhanced, environmental SEM will therefore promote Whitman's ability to continue its strong track record of student training and faculty research.

This Major Research Instrumentation award funds the acquisition of a confocal microscopy system at Whitman College. The new confocal system greatly expands Whitman College imaging capabilities, provides new research possibilities for undergraduates, and improved opportunities to integrate research into courses in Biology, Physics and our integrative Biochemistry, Biophysics, & Molecular Biology Program. Research projects that will utilize the new microscope include studies of neuronal growth and neural system development, protein crystallization, plant physiology, pollen formation, and a range of other topics. The system is also integral to a workshop co-taught by Physics and Biology professors. The confocal will be the only accessible instrument of its kind in the region, and will have a broad impact on the surrounding community through use by researchers at neighboring institutions and for demonstrations in existing Whitman outreach programs for middle school teachers, middle school and Latino high school students. Results from the studies conducted with the confocal microscope will be broadly disseminated through abstracts and peer reviewed publications, as well as by active participation of students and faculty at professional meetings.