Activity in the mouse anterior lateral motor cortex (ALM) instructs directional movements, often seconds before movement initiation. It is unknown whether this preparatory activity is localized to ALM or widely distributed within motor cortex. Here we imaged activity across motor cortex while mice performed a whisker-based object localization task with a delayed, directional licking response. During tactile sensation and the delay epoch, object location was represented in motor cortex areas that are medial and posterior relative to ALM, including vibrissal motor cortex. Preparatory activity appeared first in deep layers of ALM, seconds before the behavioral response, and remained localized to ALM until the behavioral response. Later, widely distributed neurons represented the outcome of the trial. Cortical area was more predictive of neuronal selectivity than laminar location or axonal projection target. Motor cortex therefore represents sensory, motor, and outcome information in a spatially organized manner.

Many animals maintain an internal representation of their heading as they move through their surroundings. Such a compass representation was recently discovered in a neural population in the Drosophila melanogaster central complex, a brain region implicated in spatial navigation. Here, we use two-photon calcium imaging and electrophysiology in head-fixed walking flies to identify a different neural population that conjunctively encodes heading and angular velocity, and is excited selectively by turns in either the clockwise or counterclockwise direction. We show how these mirror-symmetric turn responses combine with the neurons' connectivity to the compass neurons to create an elegant mechanism for updating the fly's heading representation when the animal turns in darkness. This mechanism, which employs recurrent loops with an angular shift, bears a resemblance to those proposed in theoretical models for rodent head direction cells. Our results provide a striking example of structure matching function for a broadly relevant computation.

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.

Close appositions between the membrane of the endoplasmic reticulum (ER) and other intracellular membranes have important functions in cell physiology. These include lipid homeostasis, regulation of Ca(2+) dynamics, and control of organelle biogenesis and dynamics. Although these membrane contacts have previously been observed in neurons, their distribution and abundance have not been systematically analyzed. Here, we have used focused ion beam-scanning electron microscopy to generate 3D reconstructions of intracellular organelles and their membrane appositions involving the ER (distance ≤30 nm) in different neuronal compartments. ER-plasma membrane (PM) contacts were particularly abundant in cell bodies, with large, flat ER cisternae apposed to the PM, sometimes with a notably narrow lumen (thin ER). Smaller ER-PM contacts occurred throughout dendrites, axons, and in axon terminals. ER contacts with mitochondria were abundant in all compartments, with the ER often forming a network that embraced mitochondria. Small focal contacts were also observed with tubulovesicular structures, likely to be endosomes, and with sparse multivesicular bodies and lysosomes found in our reconstructions. Our study provides an anatomical reference for interpreting information about interorganelle communication in neurons emerging from functional and biochemical studies.

Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes. Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain Fv (scFv) complexes, by studying a complex we failed to crystallize in pursuing an extended project of the human immune response to influenza vaccines. The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen:antibody interface, although small variation in the twist of the rod-like HA trimer limited the overall resolution to about 4.5Å. Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.

We describe new detachable floating glass micropipette electrode devices that provide targeted action potential recordings in active moving organs without requiring constant mechanical constraint or pharmacological inhibition of tissue motion. The technology is based on the concept of a glass micropipette electrode that is held firmly during cell targeting and intracellular insertion, after which a 100µg glass microelectrode, a "microdevice", is gently released to remain within the moving organ. The microdevices provide long-term recordings of action potentials, even during millimeter-scale movement of tissue in which the device is embedded. We demonstrate two different glass micropipette electrode holding and detachment designs appropriate for the heart (sharp glass microdevices for cardiac myocytes in rats, guinea pigs and humans) and the brain (patch glass microdevices for neurons in rats). We explain how microdevices enable measurements of multiple cells within a moving organ that are typically difficult with other technologies. Using sharp microdevices, action potential duration (APD) was monitored continuously for 15 minutes in unconstrained perfused hearts during global ischemia-reperfusion, providing beat-to-beat measurements of changes in APD. Action potentials from neurons in the hippocampus of anaesthetized rats were measured with patch microdevices, which provided stable base potentials during long-term recordings. Our results demonstrate that detachable microdevices are an elegant and robust tool to record electrical activity with high temporal resolution and cellular level localization without disturbing the physiological working conditions of the organ.

Advances in fluorescence microscopy promise to unlock details of biological systems with high spatiotemporal precision. These new techniques also place a heavy demand on the 'photon budget'-the number of photons one can extract from a sample. Improving the photostability of small molecule fluorophores using chemistry is a straightforward method for increasing the photon budget. Here, we review the (sometimes sparse) efforts to understand the mechanism of fluorophore photobleaching and recent advances to improve photostability through reducing the propensity for oxidation or through intramolecular triplet-state quenching. Our intent is to inspire a more thorough mechanistic investigation of photobleaching and the use of precise chemistry to improve fluorescent probes.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 10(6) µm(3). These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of Metand gce throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.