Understanding brain function requires technologies that can control the activity of large populations of neurons with high fidelity in space and time. We developed a new multiphoton holographic approach to activate or suppress the activity of ensembles of cortical neurons with cellular resolution and sub-millisecond precision. Since existing opsins were inadequate, we engineered new soma-targeted (ST) optogenetic tools, ST-ChroME and IRES-ST-eGtACR1, optimized for multiphoton activation and suppression. Employing a three-dimensional all-optical read/write interface, we demonstrate the ability to photo-stimulate up to 50 neurons simultaneously distributed in three dimensions in a 550 × 550 × 100 μm volume of brain tissue. This new approach allows the synthesis and editing of complex neural activity patterns needed to gain insight into the principles of neural codes.

During active sensation, sensors scan space in order to generate a representation of the outside world. However, since spatial coding in sensory systems is typically addressed by measuring receptive fields in a fixed, sensor-based coordinate frame, the cortical representation of scanned space is poorly understood. To address this question, we probed spatial coding in the rodent whisker system using a combination of two photon imaging and electrophysiology during active touch. We found that surround whiskers powerfully transform the cortical representation of scanned space. On the single neuron level, surround input profoundly alters response amplitude and modulates spatial preference in the cortex. On the population level, surround input organizes the spatial preference of neurons into a continuous map of the space swept out by the whiskers. These data demonstrate how spatial summation over a moving sensor array is critical to generating population codes of sensory space.

How cortical circuits build representations of complex objects is poorly understood. Individual neurons must integrate broadly over space, yet simultaneously obtain sharp tuning to specific global stimulus features. Groups of neurons identifying different global features must then assemble into a population that forms a comprehensive code for these global stimulus properties. Although the logic for how single neurons summate over their spatial inputs has been well explored in anesthetized animals, how large groups of neurons compose a flexible population code of higher-order features in awake animals is not known. To address this question, we probed the integration and population coding of higher-order stimuli in the somatosensory and visual cortices of awake mice using two-photon calcium imaging across cortical layers. We developed a novel tactile stimulator that allowed the precise measurement of spatial summation even in actively whisking mice. Using this system, we found a sparse but comprehensive population code for higher-order tactile features that depends on a heterogeneous and neuron-specific logic of spatial summation beyond the receptive field. Different somatosensory cortical neurons summed specific combinations of sensory inputs supra-linearly, but integrated other inputs sub-linearly, leading to selective responses to higher-order features. Visual cortical populations employed a nearly identical scheme to generate a comprehensive population code for contextual stimuli. These results suggest that a heterogeneous logic of input-specific supra-linear summation may represent a widespread cortical mechanism for the synthesis of sparse higher-order feature codes in neural populations. This may explain how the brain exploits the thalamocortical expansion of dimensionality to encode arbitrary complex features of sensory stimuli.

Although model organisms have provided insight into the earliest stages of cardiac vascularization, we know very little about this process in humans. Here we show that spatially micropatterned human pluripotent stem cells (hPSCs) enable in vitro modeling of this process, corresponding to the first three weeks of in vivo human development. Using four hPSC fluorescent reporter lines, we create cardiac vascular organoids (cVOs) by identifying conditions that simultaneously give rise to spatially organized and branched vascular networks within endocardial, myocardial, and epicardial cells. Using single-cell transcriptomics, we show that the cellular composition of cVOs resembles that of a 6.5 post-conception week (PCW) human heart. We find that NOTCH and BMP pathways are upregulated in cVOs, and their inhibition disrupts vascularization. Finally, using the same vascular-inducing factors to create cVOs, we produce hepatic vascular organoids (hVOs). This suggests there is a conserved developmental program for creating vasculature within different organ systems

11 How cortical circuits build representations of complex objects is poorly understood. The 12 massive dimensional expansion from the thalamus to the primary sensory cortex may enable 13 sparse, comprehensive representations of higher order features to facilitate object 14 identification. To generate such a code, cortical neurons must integrate broadly over space, yet 15 simultaneously obtain sharp tuning to specific stimulus features. The logic of cortical 16integration that may synthesize such a sparse, high dimensional code for complex features is 17 not known. To address this question, we probed the integration and population coding of 18 higher order stimuli in the somatosensory and visual cortices of awake mice using two-photon 19 calcium imaging across cortical layers. We found that somatosensory and visual cortical 20 neurons sum highly specific combinations of sensory inputs supra-linearly, but integrate other 21 inputs sub-linearly, leading to selective responses to higher order features. This integrative 22 process generates a sparse, but comprehensive code for complex stimuli from the earliest 23 stages of cortical processing. These results from multiple sensory modalities imply that input-24 specific supra-linear summation may represent a widespread cortical mechanism for the 25 synthesis of higher order feature codes. This new mechanism may explain how the brain 26