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2529 Janelia Publications
Showing 1291-1300 of 2529 resultsRats have the ability to learn and perform sophisticated behavioral tasks, making them very useful for investigating neural circuit functions. In contrast to the extensive mouse genetic toolkit, the paucity of recombinase-expressing rat models has limited the ability to monitor and manipulate molecularly-defined neural populations in this species. Here we report the generation and validation of two knock-in rat strains expressing either Cre or Flp recombinase under the control of Parvalbumin (Pvalb), a gene expressed in the critical “fast-spiking” subset of inhibitory interneurons (FSIs). These strains were generated with CRISPR-Cas9 gene editing and show highly specific and penetrant labeling of Pvalb-expressing neurons, as demonstrated by in situ hybridization and immunohistochemistry. We validated these models in both prefrontal cortex and striatum using both ex vivo and in vivo approaches, including whole-cell recording, optogenetics, extracellular physiology and photometry. Our results demonstrate the utility of these new transgenic models for a wide range of neuroscience experiments.
Hyperspectral stimulated Raman scattering microscopy is deployed to measure single-membrane vibrational spectrum as a function of membrane potential. Using erythrocyte ghost as a model, quantitative correlation between transmembrane potential and Raman spectral profile was found. Specifically, the ratio between the area under Raman band at ∼2930 cm−1 and that at ∼2850 cm−1 increased by ∼2.6 times when the potential across the erythrocyte ghost membrane varied from +10 mV to −10 mV. Our results show the feasibility of employing stimulated Raman scattering microscopy to probe the membrane potential without labeling.
The application of green-to-red photoconvertible fluorescent proteins (PCFPs) for in vivo studies in complex 3D tissue structures has remained limited because traditional near-UV photoconversion is not confined in the axial dimension, and photomodulation using axially confined, pulsed near-IR (NIR) lasers has proven inefficient. Confined primed conversion is a dual-wavelength continuous-wave (CW) illumination method that is capable of axially confined green-to-red photoconversion. Here we present a protocol to implement this technique with a commercial confocal laser-scanning microscope (CLSM); evaluate its performance on an in vitro setup; and apply primed conversion for in vivo labeling of single cells in developing zebrafish and mouse preimplantation embryos expressing the green-to-red photoconvertible protein Dendra2. The implementation requires a basic understanding of laser-scanning microscopy, and it can be performed within a single day once the required filter cube is manufactured.
The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.
L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.
The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities.
Rodents move their whiskers to locate and identify objects. Cortical areas involved in vibrissal somatosensation and sensorimotor integration include the vibrissal area of the primary motor cortex (vM1), primary somatosensory cortex (vS1; barrel cortex), and secondary somatosensory cortex (S2). We mapped local excitatory pathways in each area across all cortical layers using glutamate uncaging and laser scanning photostimulation. We analyzed these maps to derive laminar connectivity matrices describing the average strengths of pathways between individual neurons in different layers and between entire cortical layers. In vM1, the strongest projection was L2/3→L5. In vS1, strong projections were L2/3→L5 and L4→L3. L6 input and output were weak in both areas. In S2, L2/3→L5 exceeded the strength of the ascending L4→L3 projection, and local input to L6 was prominent. The most conserved pathways were L2/3→L5, and the most variable were L4→L2/3 and pathways involving L6. Local excitatory circuits in different cortical areas are organized around a prominent descending pathway from L2/3→L5, suggesting that sensory cortices are elaborations on a basic motor cortex-like plan.
The rules governing the formation of spatial maps in the hippocampus have not been determined. We investigated the large-scale structure of place field activity by recording hippocampal neurons in rats exploring a previously unencountered 48-meter-long track. Single-cell and population activities were well described by a two-parameter stochastic model. Individual neurons had their own characteristic propensity for forming fields randomly along the track, with some cells expressing many fields and many exhibiting few or none. Because of the particular distribution of propensities across cells, the number of neurons with fields scaled logarithmically with track length over a wide, ethological range. These features constrain hippocampal memory mechanisms, may allow efficient encoding of environments and experiences of vastly different extents and durations, and could reflect general principles of population coding.
We present a method combining affinity prediction with region agglomeration, which improves significantly upon the state of the art of neuron segmentation from electron microscopy (EM) in accuracy and scalability. Our method consists of a 3D U-net, trained to predict affinities between voxels, followed by iterative region agglomeration. We train using a structured loss based on MALIS, encouraging topologically correct segmentations obtained from affinity thresholding. Our extension consists of two parts: First, we present a quasi-linear method to compute the loss gradient, improving over the original quadratic algorithm. Second, we compute the gradient in two separate passes to avoid spurious gradient contributions in early training stages. Our predictions are accurate enough that simple learning-free percentile-based agglomeration outperforms more involved methods used earlier on inferior predictions. We present results on three diverse EM datasets, achieving relative improvements over previous results of 27%, 15%, and 250%. Our findings suggest that a single method can be applied to both nearly isotropic block-face EM data and anisotropic serial sectioned EM data. The runtime of our method scales linearly with the size of the volume and achieves a throughput of ~2.6 seconds per megavoxel, qualifying our method for the processing of very large datasets.