Joseph Erlichman - US grants

A grant has been awarded to Dr. Thomas W. Budd at St. Lawrence University in Canton, New York to acquire two major instruments that will complement and expand opportunities for microscopic studies and enable the creation of an Interdisciplinary, Multi-User Microscopy-Imagery Center within the science facilities complex. The instruments to be acquired are: 1. A confocal microscope system that will bridge the resolution gap between electron microscopy and fluorescence microscopy. This instrument will allow optical sectioning and 3-D reconstructions of fluorescent images with much better resolution and a wider selection of dyes than conventional fluorescence microscopes. 2. An Energy Dispersive X-Ray Analysis (EDAX) system to be fitted onto an existing scanning electron microscope (SEM). This system allows elemental analysis and spatial elemental mapping of a wide variety of research specimens applicable to biology, chemistry, environmental studies, and geology research.

Several St. Lawrence science faculty are poised to direct student projects with these instruments as part of their research programs. Dr. T. Budd (biology and PI) plans to use confocal microscopy to three-dimensionally map proteins on the surface and inside of glial-neural type cells in vitro as part of his research to characterize tissue cultured cells used to study bone formation. He will confirm the 3-D confocal results by using SEM-EDAX mapping routines. Dr. Marano (chemistry) will use confocal microscopy to detect and map the components of specialized membrane "domains" on the surface of T-lymphocytes. These domains have been implicated in gene regulation during immune responses. Dr. Erlichman (biology) will use confocal microscopy to three-dimensionally map sodium/hydrogen exchange transport systems in the surface membranes of pH sensitive neurons that regulate breathing in mammals. Dr. Erickson (geology) will use SEM-EDAX elemental analysis and mapping to study the sequence and biogeochemistry of fossilization of clams. Several other St. Lawrence faculty are also poised to use the new instruments in their research programs, all of which involve active participation by students.

This award will greatly enhance faculty and student research activity in the science departments at St. Lawrence, as well as provide instrumentation support for two new majors in biochemistry and neuroscience. As a small, private liberal arts undergraduate institution, access to state-of-the-art instrumentation is often limited. The instruments requested will enable us to better train the next generation of scientists.

DESCRIPTION (provided by applicant): Gap junctions establish pathways of intercellular communication that coordinate processes such as embryogenesis, development, growth, differentiation and cellular response to injury. In the nervous system, gap junctions establish low-resistance channels that couple cells electrically, permitting a rapid and synchronous response to stimuli. Recent work using the in vitro brainstem-spinal cord and medullary slice preparations from mice have shown that gap junction blockade decreases respiratory frequency, suggesting that gap junctions may be critical for rhythmogenesis in this preparation. Electrical coupling has been identified in several respiratory-related neuronal pools including pre-Botzinger complex, genioglossal motor neurons, phrenic motorneurons, motorneurons of the nucleus ambiguus, neurons in locus coeruleus (LC) and neurons in the nucleus tractus solitarius(NTS). Of these sites, the PBC, LC and NTS are chemosensitive and appear to be important in central respiratory control based on both in vitro and in vivo studies. Congeners of glycyrrhetinic acid (e.g. carbenoxolone) have been shown to block gap junction communication by a mechanism that may involve conformational changes in connexin structure. The role of electrical coupling in central chemosensory function in vivo has not been examined. Here we show that unilateral, focal stimulation of the rostral NTS leads to a significant increase in ventilation in the conscious adult rat. Pharmacological blockade of gap junctions in the NTS with carbenoxolone decreased ventilation in young adults (about 7 weeks old) but not in older adults (about 14 weeks old). These findings suggest that electrical coupling at some sites involved in central respiratory control may give way to chemical synaptic transmission as the animal develops. In support of this hypothesis, we show that unilateral, focal perfusion of cobalt into the rostral NTS decreases ventilation about 60%, suggesting an important role of chemical synaptic transmission in the older adult. In this proposal we will examine 3 putative chemoreceptor sites. Two of these sites demonstrate electrical coupling between cells (the NTS and the LC) whereas the third chemosensitive site does not (the retrotrapezoid nucleus). Here we outline experiments designed to examine the changes in the relative contribution of electrical and chemical synaptic transmission in the NTS during development using the chronically instrumented, conscious rats.

Glial cells have been implicated in diverse functions including the metabolic support of neurons and maintenance of extracellular ion concentrations. Recent evidence suggests that glia may play an important role in regulating the extracellular concentrations of neurotransmitters, potassium ions and hydrogen ions (protons). Acidosis (elevation of proton levels) can occur when respiratory activity is low. Acidosis stimulates neural cells within the brainstem that respond to elevated CO2 concentrations. The changes in neuronal electrical activity of these cells are determined in part by the extracellular pH (acid levels). Therefore, factors that influence pH in or around these chemosensitive cells may affect ventilatory responsiveness to acidic stimuli.

The proposed work is anticipated to yield the following important findings: 1) identify pH regulating mechanisms present in brainstem glia; 2) determine the extent to which extracellular pH (pHo) regulation depends on the activation of proton transport in glia; and 3) examine how selective blocking of glial pH regulation can affect pHo and, in turn, affect pHi (the pH within the cells) and the chemosensory stimulus in CO2-sensitive brainstem neurons.

Undergraduate students participating in the proposed research will be fully immersed in all aspects of experimental design, data collection, analysis and interpretation thereby providing opportunities for the students to explore their own ingenuity and creativity within the context of a scientifically relevant problem. One of the primary goals of this faculty-initiated, student-driven research is to establish a mentoring partnership between the student and faculty member that fosters the student's understanding of the process of scientific inquiry and the refinement of scientific questions through experimentation. The students will gain a clear understanding of the process of doing science and consequently hone many life skills (e.g. critical thinking, oral, visual and written communication) that will serve them well throughout their careers.

0923398
Chiarenzelli

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

Funds from this grant will support the acquisition of a scanning electron microscope (SEM) equipped with a variable temperature sample stage, cathodoluminescence (CL) and energy dispersive spectrometers (EDS). The SEM will support the research and teaching activities at least nine faculty members across the disciplines of geology, biology, and physics at Saint Lawrence University, an undergraduate institution. Two of the PIs are new to the NSF proposal process. The SEM will serve the needs of a broad group of productive scientists for applications in tectonics /geochronology, identification of fossils in marine carbonates, research in neuroscience, and characterization of novel ceramics. Undergraduate student access to an on campus state-of-the-art SEM will facilitate student engagement in modern research methods and better prepare them for careers in numerous high tech industries that rely on high resolution microscopy for materials characterization.

An award is made to St. Lawrence University (SLU) to acquire a confocal microscopy system. Many modern cellular and molecular biology techniques use natural fluorescence, fluorescently tagged molecules, or fluorescent dyes to label specific cellular components. A confocal microscope is an instrument that uses lasers to produce high resolution images of cellular and subcellular components within living and fixed cells that have been labeled with one or more fluorescent molecules. In addition, 3D reconstructions of these fluorescent structures can be created using a series of images collected by the confocal microscope as it focuses down through a specimen. Consequently, this instrument has the resolving power to address many types of biological problems including identifying cell types, cellular localization of specific molecules, determining gene expression, and examining cell differentiation. The research faculty involved in this project require the enhanced flexibility of use and special capabilities (e.g., UV laser, spectral detector, and environmental chamber) of this confocal system to obtain quality images of their work for their contributions to their scientific fields, including publications, national and international meeting presentations, student training, and other applications. Thus, this award will broaden research, training, and teaching opportunities both at SLU and the Associated Colleges of the St. Lawrence Valley Consortium. Acquisition of this instrument will expand the existing partnership between SLU and Clarkson University (CU), especially increasing summer fellowship projects for CSTEP and McNair Scholars; two programs that support students from populations underrepresented in STEM. In addition, the new microscope will fuel outreach activities for dozens of K-12 teachers, students, and community members in our economically disadvantaged, rural region of northern New York.

A Nikon C2+ spectral imaging confocal microscope system with wide field camera and environmental chamber systems and specialized capabilities will support and expand current and future research, teaching, and undergraduate training activities of STEM faculty and students at the Associated Colleges of the St. Lawrence Valley (SLU, Clarkson, SUNY Potsdam, and SUNY Canton), a higher education consortium in upstate New York. The new instrument will augment the research and scholarly contributions of research faculty and enhance teaching and training activities of eleven faculty and science professionals in cell and developmental biology and ecology and evolution, including the PI and Co-PIs' projects, which focus on the theme of subcellular trafficking and tissue localization of specific molecules during development. The acquisition of this confocal microscope will provide the flexibility important for all users to achieve successful imaging with their respective biological systems of study, while also being easy to use and having a relatively low cost infrastructure. In particular, this system will allow research faculty to explore the biological effects of cerium oxide nanoparticles; gene expression in cheilostome bryozoans; and the essential role of the evolutionarily conserved IME4 mRNA methyltransferase in metazoan development. High-resolution imaging is required to publish in high profile cell and molecular biology journals, and confocal microscopy is now the expected minimal standard for publishing microscopy-based research using fluorescence. Specifically, this instrument will positively impact the relatively new field of cerium oxide nanoparticles, as the new system will enable Drs. Erlichman and Estevez to characterize how the chemistry of cerium oxide nanoparticles influences their cellular trafficking and whether cellular localization influences pro-oxidant vs antioxidant effects. Body axes are determined multiple times during the bryozoan life cycle (embryogenesis, metamorphosis, and asexual budding). Using the proposed instrument, Dr. Temkin will acquire data on Hox gene expression during two different patterns of asexual budding. These data will provide a foundation to examine Hox gene expression at other points in the bryozoan life cycle. Lastly, a fundamental question in developmental biology is how cell fates are determined. To answer this, Dr. Hongay will employ the new microscope to perform high resolution confocal analyses to determine how a highly evolutionarily conserved RNA-modifying enzyme dictates cell fates and cell differentiation in Drosophila spermatogenesis.