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  1. Abstract

    Spatial cognition depends on an accurate representation of orientation within an environment. Head direction cells in distributed brain regions receive a range of sensory inputs, but visual input is particularly important for aligning their responses to environmental landmarks. To investigate how population-level heading responses are aligned to visual input, we recorded from retrosplenial cortex (RSC) of head-fixed mice in a moving environment using two-photon calcium imaging. We show that RSC neurons are tuned to the animal’s relative orientation in the environment, even in the absence of head movement. Next, we found that RSC receives functionally distinct projections from visual and thalamic areas and contains several functional classes of neurons. While some functional classes mirror RSC inputs, a newly discovered class coregisters visual and thalamic signals. Finally, decoding analyses reveal unique contributions to heading from each class. Our results suggest an RSC circuit for anchoring heading representations to environmental visual landmarks.

     
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  2. Abstract

    Understanding brain function requires monitoring local and global brain dynamics. Two-photon imaging of the brain across mesoscopic scales has presented trade-offs between imaging area and acquisition speed. We describe a flexible cellular resolution two-photon microscope capable of simultaneous video rate acquisition of four independently targetable brain regions spanning an approximate five-millimeter field of view. With this system, we demonstrate the ability to measure calcium activity across mouse sensorimotor cortex at behaviorally relevant timescales.

     
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  3. Abstract

    Visual cortical responses are known to be highly variable across trials within an experimental session. However, the long-term stability of visual cortical responses is poorly understood. Here using chronic imaging of V1 in mice we show that neural responses to repeated natural movie clips are unstable across weeks. Individual neuronal responses consist of sparse episodic activity which are stable in time but unstable in gain across weeks. Further, we find that the individual episode, instead of neuron, serves as the basic unit of the week-to-week fluctuation. To investigate how population activity encodes the stimulus, we extract a stable one-dimensional representation of the time in the natural movie, using an unsupervised method. Most week-to-week fluctuation is perpendicular to the stimulus encoding direction, thus leaving the stimulus representation largely unaffected. We propose that precise episodic activity with coordinated gain changes are keys to maintain a stable stimulus representation in V1.

     
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  4. Abstract

    To produce consistent sensory perception, neurons must maintain stable representations of sensory input. However, neurons in many regions exhibit progressive drift across days. Longitudinal studies have found stable responses to artificial stimuli across sessions in visual areas, but it is unclear whether this stability extends to naturalistic stimuli. We performed chronic 2-photon imaging of mouse V1 populations to directly compare the representational stability of artificial versus naturalistic visual stimuli over weeks. Responses to gratings were highly stable across sessions. However, neural responses to naturalistic movies exhibited progressive representational drift across sessions. Differential drift was present across cortical layers, in inhibitory interneurons, and could not be explained by differential response strength or higher order stimulus statistics. However, representational drift was accompanied by similar differential changes in local population correlation structure. These results suggest representational stability in V1 is stimulus-dependent and may relate to differences in preexisting circuit architecture of co-tuned neurons.

     
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  5. Abstract

    Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long‐term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage‐gated luciferase coupling the functionalities of the Ciona voltage‐sensing domain (VSD) and bacterial luciferase, luxAB. When co‐expressed with the luciferin‐producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several‐fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons ofCaenorhabditis elegansis expressed. Using the compressed sensing approach, the electropharingeogram of theC. eleganspharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.

     
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  6. Abstract

    Perception of visual motion is important for a range of ethological behaviors in mammals. In primates, specific visual cortical regions are specialized for processing of coherent visual motion. However, whether mouse visual cortex has a similar organization remains unclear, despite powerful genetic tools available for measuring population neural activity. Here, we use widefield and 2-photon calcium imaging of transgenic mice to measure mesoscale and cellular responses to coherent motion. Imaging of primary visual cortex (V1) and higher visual areas (HVAs) during presentation of natural movies and random dot kinematograms (RDKs) reveals varied responsiveness to coherent motion, with stronger responses in dorsal stream areas compared to ventral stream areas. Moreover, there is considerable anisotropy within visual areas, such that neurons representing the lower visual field are more responsive to coherent motion. These results indicate that processing of visual motion in mouse cortex is distributed heterogeneously both across and within visual areas.

     
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  7. Abstract

    Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~ 450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus in rodents. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration, particularly in non-rodent species. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500–600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5–6 and subsequent three-photon imaging of orientation- and direction- selective visual responses from these cells.

     
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  8. While two-photon fluorescence microscopy is a powerful platform for the study of functional dynamics in living cells and tissues, the bulk motion inherent to these applications causes distortions. We have designed a motion tracking module based on spectral domain optical coherence tomography which compliments a laser scanning two-photon microscope with real-time corrective feedback. The module can be added to fluorescent imaging microscopes using a single dichroic and without additional contrast agents. We demonstrate that the system can track lateral displacements as large as 10μm at 5 Hz with latency under 14 ms and propose a scheme to extend the system to 3D correction with the addition of a remote focusing module. We also propose several ways to improve the module’s performance by reducing the feedback latency. We anticipate that this design can be adapted to other imaging modalities, enabling the study of samples subject to motion artifacts at higher resolution.

     
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  9. Spatial frequency modulation for imaging (SPIFI) has traditionally employed a time-varying spatial modulation of the excitation beam. Here, for the first time to our knowledge, we introduce single-shot SPIFI, where the spatial frequency modulation is imposed across the entire spatial bandwidth of the optical system simultaneously enabling single-shot operation.

     
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  10. Imaging beyond the diffraction limit barrier has attracted wide attention due to the ability to resolve previously hidden image features. Of the various super-resolution microscopy techniques available, a particularly simple method called saturated excitation microscopy (SAX) requires only simple modification of a laser scanning microscope: The illumination beam power is sinusoidally modulated and driven into saturation. SAX images are extracted from the harmonics of the modulation frequency and exhibit improved spatial resolution. Unfortunately, this elegant strategy is hindered by the incursion of shot noise that prevents high-resolution imaging in many realistic scenarios. Here, we demonstrate a technique for super-resolution imaging that we call computational saturated absorption (CSA) in which a joint deconvolution is applied to a set of images with diversity in spatial frequency support among the point spread functions (PSFs) used in the image formation with saturated laser scanning fluorescence microscopy. CSA microscopy allows access to the high spatial frequency diversity in a set of saturated effective PSFs, while avoiding image degradation from shot noise.

     
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