A comprehensive understanding of brain cell diversity and cell-to-cell communication (connectivity via specialized junctions between cells called synapses) is essential for understanding the anatomical substrates and cellular mechanisms that underlie brain function. Elucidation of the detailed synaptic organization of specific neural circuits is a necessary component for achieving this goal. Progress in this area of neuroscience has been limited by a lack of tools that can simultaneously reveal multiple cell types with distinct molecular and physiological properties within the same brain areas, as well as highlighting in detail how these cells are connected to each other. This project utilizes a novel molecular-biological strategy for defining multiple cell types and their interconnections in mouse brains, enabling investigators to mark individual cells that express combinations of engineered genetic elements. This technique is combined with an existing method that inserts two incomplete parts of a fluorescent molecule onto the surfaces of different nerve cells. If those cells communicate by forming a connection (synapse) with each other, the fragments of the fluorescent molecule come into close enough contact to become functional again, and synapses are identifiable as colored dots of fluorescent light. Both graduate and undergraduate students will be involved in the development and optimization of these tools, and the genetic constructs will be distributed via a non-profit agency; all protocols and sequence information will be made widely available through the PI's website. These new tools will greatly enhance the speed and precision with which neuroscientists can study the detailed organization of brain circuitry, and the synaptic connections made onto specific neurons.

This project introduces novel molecular and viral strategies for defining multiple cell types and their synaptic organization. It is based on a novel viral strategy to achieve the intersectional expression of transgenes among specific neuronal populations in transgenic mouse lines that express Cre, flippase (FLP), and other recombinases. A novel site-specific recombinase system (phiC31 and a phiC31-dependent single inverted open reading frame [pSIO]) is used to acheive virus-mediated, specific-transgene expression without cross-reactivity to other recombinases, both in vitro and in vivo. This phiC31/pSIO recombinase system is then combined with "enhanced Green fluorescent protein Reconstitution Across Synaptic Partners" (eGRASP) to label synaptic contacts made by specific inputs. By providing a novel experimental framework for identifying the cellular constituents of local neural circuits and examining the synaptic organization of multiple presynaptic inputs onto specific neurons, and by optimizing these tools for general use and making them freely available to the scientific community, this project is expected to accelerate discovery about the organizational and functional properties of neural circuitry.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Evan Balaban
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University of California San Diego
La Jolla
United States
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