Thomas Mrsic-Flogel - US grants

To understand brain function in health and disease it is essential to rapidly monitor signaling in neural circuits. Two-photon microscopy is a core tool for neuroscience research because it enables neuronal activity to be monitored at high spatial resolution deep within brain tissue. However, the mechanical scanning and focusing of conventional designs severely limits the temporal resolution of 30 imaging and brain movement complicates recordings. We have developed a novel compact acousto-optic lens (AOL) 3D two-photon microscope that overcomes these limitations, allowing high speed imaging over volumes spanning hundreds of micrometres and real time correction of the brain movement that occurs in awake behaving animals. The agile 3D random access pointing and scanning (RAPS) at 20-40 kHz bridges the gap in the temporal resolution between optical imaging and electrophysiology. Moreover, compact design features of our AOL enable it to be added to existing two-photon microscopes at relatively low cost. This proposal aims to refine and rapidly disseminate this powerful new technology. Technology refinements include expanding the volumes that can be imaged and extending AOL-based real-time movement correction from 20 to 30. The close collaboration with experimentalists in our lab and other key leading groups distributed worldwide (to whom the technology will be initially disseminated) will ensure that current and new features of the AOL, as well as the open source GUI software, are robust and meet the requirements of the neuroscience community. This will deliver new, urgently needed technological developments required for investigating neural circuits and accelerate dissemination of this world leading technology well ahead of the 3 year timescale that is required by commercial manufacturers to bring a product to market.

Summary/Abstract, Core C: Histology The overall goal of this U19 application is to determine the brainwide neural basis and behavioral consequences of internal states, such as task engagement, as mice perform a standardized decision-making task. Registration of electrophysiological recording sites into a common anatomical framework represents a critical step in comparing these experiments across projects and synthesizing the results into a theoretical model, so an accurate and robust histological and image analysis methodology is essential for the development of high-quality, reproducible datasets. Histology Core C therefore aims to deliver two key outcomes. First, this core will centralize and standardize histology data acquisition and registration to a reference atlas across experimental Projects 1, 2, and 4. Through previous work by groups participating in this proposal, the anatomical reconstruction and registration pipeline for recordings with Neuropixels probes using serial-section two-photon imaging has been established, optimized, and validated. This core will develop an open-source software package for sample registration to the Allen mouse brain common coordinate framework, version 3 (CCF). This package will include functions for assessing registration quality, and will be extended to handle new experimental recording methods as needed for the proposed experiments, including the reconstruction and registration of labeled cells investigated with calcium imaging, the registration of functional ultrasound imaging data onto the Allen CCF, and the reconstruction of cells, pathways, and optic fiber insertion sites in fiber photometry and optogenetic stimulation experiments. Second, to support the additional experimental aims proposed in this application, particularly optogenetic perturbations in Project 2 and calcium imaging in Project 4, this core will develop clearing and histological labeling protocols to interrogate nervous system structure. Many of the technical hurdles to delivering these aims have already been overcome. The centralizing of histological processing in the International Brain Laboratory consortium is well established, and a first version of this open-source software is in its final stages of development. A whole-brain tissue clearing and lightsheet imaging methodology has been demonstrated, and its reliable registration to the common framework is under assessment. A range of histological procedures that can immunohistochemically label large blocks of tissue are available, and will form the basis for further histological interrogation. In conclusion, Histology Core C will perform the essential tasks of processing mouse brains for histological reconstruction, generating structural datasets from this tissue in close collaboration with U19 projects, and supplying the resulting protocols, software tools, and datasets to the wider community.