Probing functional connectivity in vivo via holographic and molecular targeting

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The relationship between the function and connectivity of cortical circuits has been a long-standing theme in neuroscience. Although since decades the predominant pathways of cortical information processing in primary sensory area have been described, little is known about how stimulus selectivity is constructed by the number and strength of synaptic connections within and between cortical laminae. Recent evidences indicate a correlation between the weight of horizontal excitatory connections and stimulus preference. The classical approach of inserting multiple microelectrodes to record and map connectivity cannot be applied for tens to hundreds of neurons in vivo. In the proposed project, we aim at uncovering the connectivity patterns underlying visual processing in mouse V1 by harnessing the complementary expertise from the two partner teams, i.e., wavefront shaping methods (Emiliani Lab) and engineering of optogenetic actuators (Hegemann Lab).Our holographic light-patterning approach has proved its capability of millisecond control of action potential (AP) generation via two-photon (2P) excitation in vitro and in vivo. The use of soma-targeted opsin ensures unbiased single-cell activation. To extend millisecond AP induction to target neurons situated at layer L2/3, L4, and L5 in vivo, we will tailor optical and molecular parameters for 2P imaging and 2P stimulation up to ~1 mm deep below the brain surface. To assess an opsin/reporter combination of minimum crosstalk activation, we will test custom-designed opsins of different absorption spectra, channel kinetics, and membrane targeting to be combined with calcium or voltage indicators in an optical system based on our latest scheme of 3D light-shaping. To overcome the light intensity loss due to tissue scattering for deep imaging and deep stimulation, on the one hand we will optimize optical strategies of underfilling objective pupil, temporal focusing, and structured light-patterning, and on the other hand we will test the benefits of red-shifted opsins or reporters.In contrast to correlating orientation selectivity determination in vivo with connectivity identified post hoc in vitro, here we set out to probe the functional wiring all in vivo with optical approaches. We will map monosynaptic connections via holographic illumination onto a presynaptic neuron expressing soma-targeted opsin while monitoring the postsynaptic excitatory potential (EPSP) via a patch electrode. By sequentially moving the holographic spots onto neurons across cortical layers, we will be able to identify both horizontal and vertical connections relative to the postsynaptic cell. The main advantage is that our holographic method enables millisecond generation of single AP in a presynaptic neuron, thus allowing determining monosynaptic connection based on EPSP latency. Furthermore, activity readout via voltage indicator will permit all-optical mapping the directionality of neuronal connections.