PROJECT SUMMARY Understanding information flow in cortical circuits requires understanding both the anatomical connectivity between neurons and the way in which inputs to a neuron are integrated to generate a spiking output. Many techniques are now available to study connectivity across cells and brain areas, but the dendritic integration of these inputs is challenging to observe because we lack access to the complex electrical signals in fine dendrites. The potential for fluorescent voltage sensors to revolutionize our understanding of the functional role of dendritic integration in cortical circuits has been recognized for decades. However, in practice, fluorescent voltage sensors have lacked the necessary characteristics in terms of brightness, sensitivity, and photostability to enable their use in the challenging application of dendritic imaging in vivo. Furthermore, many fluorescent voltage sensors are unsuitable for two-photon imaging, which is required to resolve dendrites at the scale of individual branches and spines hundreds of microns deep in intact tissue. A new generation of genetically-encoded “JEDI” sensors developed in the St. Pierre lab here at BCM overcome many of these limitations, and have now been validated for two-photon somatic imaging in vivo. These validation experiments suggest that JEDI sensors have the necessary sensitivity, photostability, and brightness to enable imaging of electrical activity in dendrites in vivo. Complementing the development of these sensors, technologies for two-photon imaging of dendrites, including adaptive optics for increasing spatial resolution and Bessel beam shaping for imaging sparsely-labeled dendrites have been developed by the Ji lab at Berkeley. These techniques have been validated with dendritic calcium imaging, but have not been combined with JEDI voltage sensors in dendrites in vivo. In this R34 proposal, we will perform a careful series of validation measurements that will enable us to optimize advanced two-photon imaging of JEDI voltage sensors for interrogating dendritic electrophysiology. These experiments will unambiguously reveal the extent to which this approach has sufficient temporal and spatial resolution to enable observations of key aspects of dendritic integration in vivo. Optimizing these techniques will be widely valuable for the field, and will enable a future BRAIN Circuits project to answer fundamental questions about how pyramidal neurons in primary visual cortex circuits integrate different sources of visual information in their dendritic arbors, and how this process of integration is shaped by neuromodulators such as acetylcholine across different brain states.