INTERNATIONAL GUEST SPEAKER - 14TH ANNUAL BURDON SANDERSON CARDIAC SCIENCE LECTURE: Optogenetic, tissue clearing, and viral vector approaches to understand and influence whole-animal physiology and behaviour

Our research group at Caltech develops and employs optogenetics, tissue clearing, and viral vectors to gain new insights on circuits underlying locomotion, reward, and sleep. In particular we will discuss how bidirectional manipulation of mesopontine cholinergic cell bodies exerted opposing effects on locomotor behavior and reinforcement learning and how these effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral substantia nigra pars compacta or to the ventral tegmental area, respectively (Xiao et al, Neuron, 2016). Genetically encoded tools that can be used to visualize, monitor, and modulate mammalian neurons are revolutionizing neuroscience. However, use of genetic tools in non-transgenic animals is often hindered by the lack of vectors capable of safe, efficient, and specific delivery to the desired cellular targets. To begin to address these challenges, we have developed an in vivo Cre-based selection platform (CREATE) for identifying adeno-associated viruses (AAVs) that more efficiently transduce genetically defined cell populations (Deverman et al, Nature Biotechnology, 2016). As a first test of the CREATE platform, we selected for viruses that transduced the brain after intravascular delivery and found a novel vector, AAV-PHP.B, that transduces most neuronal types and glia across the brain. We also demonstrate how whole-body tissue clearing can facilitate transduction maps of systemically delivered genes (Yang et al, Cell, 2014; Treweek et al, Nature Protocols, 2016) and how non-invasive delivery vectors can be used to achieve dense to sparse labeling to enable morphology tracing (unpublished). Since CNS disorders are notoriously challenging due to the restrictive nature of the blood brain barrier, the recombinant vectors engineered to overcome this barrier can enable potential future use of exciting advances in gene editing via the CRISPR-Cas, RNA interference and gene replacement strategies to restore diseased CNS circuits. In addition to control of neuronal activity we need feedback on how exactly the tissue is responding to modulation. We have worked on two related topics: optical voltage sensors and imaging of single molecule RNA in cleared tissue. We used directed evolution of opsins to make them better at reporting action potentials (Flytzanis et al, Nature Communications, 2014). Changes in RNA transcripts can also report on activity history of brain circuits. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas, from developmental biology to neuroscience. Collaborators and us recently reported on methods for multi-color, multi-RNA, imaging in deep tissues. By using single-molecule hybridization chain reaction (smHCR), PACT tissue hydrogel embedding and clearing and light-sheet microscopy we detected single-molecule mRNAs in ~mm-thick brain tissue samples (Shah et al, Development, 2016) and by rRNA labeling we mapped the identity and growth rate of pathogens in clinical samples (DePas et al, mBio, 2016). Together these technologies can enable high content anatomical and functional mapping to define changes that affect cell function and health body-wide.