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Research
The Topics - Neural Circuit Formation, Learning, Memory and Sleep
Neural circuit formation, learning, memory and the role of sleep on the nervous system remain poorly understood despite their profound importance in normal cognition, and their emerging role in devastating neurological disorders, such as schizophrenia and dementia.
We are discovering new and unexpected pathways that mediate critical neurophysiological processes using a suite of powerful tools, including fluorescent biomarkers that we developed to visualize specific neural connections called Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) and NLG-1 Cherry Linker Across Synaptic Partners (NLG-1 CLASP). In addition, we use biomarkers to cell-specifically visualize neuronal calcium and cGMP signaling, and circuit-specific behavioral assays combined with molecular and genetic approaches to elucidate novel regulatory pathways. We focus on C. elegans, a nematode whose nervous system resembles that of humans at the molecular and cellular levels.
We are currently working on three NIH-funded major projects, two of which are collaborations with researchers at the University of California, San Francisco. Our research is highly collaborative, and has recently involved publications with faculty in mathematics, chemistry, computer science and physics.
The Model Organism - C. elegans
We chose to study these topics in the microscopic nematode (roundworm) C. elegans because it is an ideal model organism for genetic studies due to the extensive genetic and molecular tools available. Cell specific promoters have been characterized which allow the study of interactions between individual neurons. In addition, previous work indicates that synaptic molecules and molecules that regulate sleep in humans are conserved in C. elegans. Most importantly, it is the only organism for which a complete map of synaptic connections has been generated through decades of study, making it ideal for the synaptic study.
Our Tools - Utilizing NLG-1 GRASP and NLG-1 CLASP to visualize specific synapses in live animals
Most synapses exist in complex environments with multiple neurons found in compact regions. Visualizing alignment of existing pre- and postsynaptic markers is frequently not possible in such complex environments due to the resolution limit of conventional light microscopy. Reconstruction of synaptic connections using electron microscopy would take months to years for each animal, making it impractical for genetic and behavioral studies.
Figure 1. NLG-1 GRASP and NLG-1 CLASP are novel transgenic markers that label correct synaptic partner recognition with green and sfCherry fluorescence respectively.
To address these questions, we developed novel trans-synaptic markers called NLG-1 GRASP and NLG-1 CLASP that allow visualization of changes in synaptic connectivity in live animals with conventional fluorescence microscopy. We fused complementary fragments of a split GFP (Green Fluorescent Protein) or split sfCherry to Neuroligin 1, which we found to localize both pre- and postsynaptically. These systems offer simple and rapid means to query specific synapses: the presence (or absence) of fluorescence indicates the formation (or lack) of the appropriate synapses.
Using NLG-1 GRASP and NLG-1 CLASP, we have successfully visualized changes in connectivity in multiple characterized circuits using known synaptic specificity mutants in live animals. We are now taking genetic approaches to discover novel molecular mechanisms guiding synaptic partner recognition in complex nerve bundles, broad sleep-dependent synaptic changes, and synaptic changes during learning and memory.