Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

/pdf/ultrasensitive-fluorescent-proteins-for-imaging-neuronal-1f4lmx12cf.pdf

Ultrasensitive fluorescent proteins for imaging neuronal activity.

http://pubs.acs.org/doi/pdf/10.1021/cr400546e

Fluorescent Sensors for Measuring Metal Ions in Living Systems

Optogenetics in neural systems.

Genetically encoded, single-component optogenetic tools have made a significant impact on neuroscience, enabling specific modulation of selected cells within complex neural tissues. As the optogenetic toolbox contents grow and diversify, the opportunities for neuroscience continue to grow. In this review, we outline the development of currently available single-component optogenetic tools and summarize the application of various optogenetic tools in diverse model organisms.

/pdf/the-development-and-application-of-optogenetics-49rsxufk6s.pdf

The Development and Application of Optogenetics

The physical linkage between the extracellular matrix and the actin cytoskeleton of a cell is made by structures known as focal adhesions, acting through integrin receptors. They are of fundamental importance in human physiology because they mediate cell adhesion, mechanosensing and signalling for the control of cell growth and differentiation. The molecular architecture of focal adhesions has now been determined using three-dimensional super-resolution fluorescence microscopy to map protein organization at the nanoscale level. They are revealed as well-organized ultrastructures in which integrins and actin are separated by a 40-nanometre-long core consisting of partially overlapping protein-specific layers, spanned by talin tethers. The multilaminar architecture creates three or more separate compartments that mediate the interdependent functions of focal adhesions. Focal adhesions link the extracellular matrix by integrin receptors to cytoplasmic actin filaments and are fundamental to human physiology. These authors determine the molecular architecture of focal adhesions by mapping protein organization at the nanoscale level. The results demonstrate that focal adhesions possess a well-organized ultrastructure made up of at least three spatial and functional compartments that mediate their interdependent functions. Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing1,2. Focal adhesions are multifunctional organelles that mediate cell–ECM adhesion, force transmission, cytoskeletal regulation and signalling1,2,3. Focal adhesions consist of a complex network4 of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque5,6 linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine7,8. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy)9 to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ∼40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin’s polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

/pdf/nanoscale-architecture-of-integrin-based-cell-adhesions-ulyvge6gol.pdf

Nanoscale architecture of integrin-based cell adhesions

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

/pdf/imaging-neural-activity-in-worms-flies-and-mice-with-3p3ty3yt7l.pdf

Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators

Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the approximately 10-nm localization precision of its parent.

Sean A McKinney

Papers

Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators

A bright and photostable photoconvertible fluorescent protein