Imaging cortical dynamics in GCaMP transgenic rats with a head-mounted widefield macroscope

Widefield imaging of calcium dynamics is an emerging method for mapping regional neural activity but is currently limited to restrained animals. Here we describe cScope, a head- mounted widefield macroscope developed to image large-scale cortical dynamics in rats during natural behavior. cScope provides a 7.8 by 4 mm field of view, dual illumination paths for both fluorescence and hemodynamic correction, and can be fabricated at low cost using readily attainable components. We also report the development of Thy-1 transgenic rat strains with widespread neuronal expression of the calcium indicator GCaMP6f. We combined these two technologies to image large-scale calcium dynamics in the dorsal neocortex during a visual evidence accumulation task. Quantitative analysis of task-related dynamics revealed multiple regions having neural signals that encode behavioral choice and sensory evidence. Our results provide a new transgenic resource for calcium imaging in rats and extend the domain of headmounted microscopes to larger-scale cortical dynamics.Accepted manuscrip

Shared Song Detector Neurons in Drosophila Male and Female Brains Drive Sex-Specific Behaviors

Males and females often produce distinct responses to the same sensory stimuli. How such differences arise-at the level of sensory processing or in the circuits that generate behavior-remains largely unresolved across sensory modalities. We address this issue in the acoustic communication system of Drosophila. During courtship, males generate time-varying songs, and each sex responds with specific behaviors. We characterize male and female behavioral tuning for all aspects of song and show that feature tuning is similar between sexes, suggesting sex-shared song detectors drive divergent behaviors. We then identify higher-order neurons in the Drosophila brain, called pC2, that are tuned for multiple temporal aspects of one mode of the male's song and drive sex-specific behaviors. We thus uncover neurons that are specifically tuned to an acoustic communication signal and that reside at the sensory-motor interface, flexibly linking auditory perception with sex-specific behavioral responses

Germ Plasm Anchoring Is a Dynamic State that Requires Persistent Trafficking

Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP) particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, we show that RNP particle motility is required for long-term germ plasm retention. We propose that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations

Volume conservation principle involved in cell lengthening and nucleus movement during tissue morphogenesis

Tissue morphogenesis is the process in which coordinated movements and shape changes of large numbers of cells form tissues, organs, and the internal body structure. Understanding morphogenetic movements requires precise measurements of whole-cell shape changes over time. Tissue folding and invagination are thought to be facilitated by apical constriction, but the mechanism by which changes near the apical cell surface affect changes along the entire apical–basal axis of the cell remains elusive. Here, we developed Embryo Development Geometry Explorer, an approach for quantifying rapid whole-cell shape changes over time, and we combined it with deep-tissue time-lapse imaging based on fast two-photon microscopy to study Drosophila ventral furrow formation. We found that both the cell lengthening along the apical–basal axis and the movement of the nucleus to the basal side proceeded stepwise and were correlated with apical constriction. Moreover, cell volume lost apically due to constriction largely balanced the volume gained basally by cell lengthening. The volume above the nucleus was conserved during its basal movement. Both apical volume loss and cell lengthening were absent in mutants showing deficits in the contractile cytoskeleton underlying apical constriction. We conclude that a single mechanical mechanism involving volume conservation and apical constriction-induced basal movement of cytoplasm accounts quantitatively for the cell shape changes and the nucleus movement in Drosophila ventral furrow formation. Our study provides a comprehensive quantitative analysis of the fast dynamics of whole-cell shape changes during tissue folding and points to a simplified model for Drosophila gastrulation

Zebrafish mutations affecting cilia motility share similar cystic phenotypes and suggest a mechanism of cyst formation that differs from pkd2 morphants.

Zebrafish are an attractive model for studying the earliest cellular defects occurring during renal cyst formation because its kidney (the pronephros) is simple and genes that cause cystic kidney diseases (CKD) in humans, cause pronephric dilations in zebrafish. By comparing phenotypes in three different mutants, locke, swt and kurly, we find that dilations occur prior to 48 hpf in the medial tubules, a location similar to where cysts form in some mammalian diseases. We demonstrate that the first observable phenotypes associated with dilation include cilia motility and luminal remodeling defects. Importantly, we show that some phenotypes common to human CKD, such as an increased number of cells, are secondary consequences of dilation. Despite having differences in cilia motility, locke, swt and kurly share similar cystic phenotypes, suggesting that they function in a common pathway. To begin to understand the molecular mechanisms involved in cyst formation, we have cloned the swt mutation and find that it encodes a novel leucine rich repeat containing protein (LRRC50), which is thought to function in correct dynein assembly in cilia. Finally, we show that knock-down of polycystic kidney disease 2 (pkd2) specifically causes glomerular cysts and does not affect cilia motility, suggesting multiple mechanisms exist for cyst formation

Cellular Mechanisms of Alpha Herpesvirus Egress: Live Cell Fluorescence Microscopy of Pseudorabies Virus Exocytosis

<div><p>Egress of newly assembled herpesvirus particles from infected cells is a highly dynamic process involving the host secretory pathway working in concert with viral components. To elucidate the location, dynamics, and molecular mechanisms of alpha herpesvirus egress, we developed a live-cell fluorescence microscopy method to visualize the final transport and exocytosis of pseudorabies virus (PRV) particles in non-polarized epithelial cells. This method is based on total internal reflection fluorescence (TIRF) microscopy to selectively image fluorescent virus particles near the plasma membrane, and takes advantage of a virus-encoded pH-sensitive probe to visualize the precise moment and location of particle exocytosis. We performed single-particle tracking and mean squared displacement analysis to characterize particle motion, and imaged a panel of cellular proteins to identify those spatially and dynamically associated with viral exocytosis. Based on our data, individual virus particles travel to the plasma membrane inside small, acidified secretory vesicles. Rab GTPases, Rab6a, Rab8a, and Rab11a, key regulators of the plasma membrane-directed secretory pathway, are present on the virus secretory vesicle. These vesicles undergo fast, directional transport directly to the site of exocytosis, which is most frequently near patches of LL5β, part of a complex that anchors microtubules to the plasma membrane. Vesicles are tightly docked at the site of exocytosis for several seconds, and membrane fusion occurs, displacing the virion a small distance across the plasma membrane. After exocytosis, particles remain tightly confined on the outer cell surface. Based on recent reports in the cell biological and alpha herpesvirus literature, combined with our spatial and dynamic data on viral egress, we propose an integrated model that links together the intracellular transport pathways and exocytosis mechanisms that mediate alpha herpesvirus egress.</p></div

Volumetric two-photon imaging of neurons using stereoscopy (vTwINS)

Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large scale recording of neural activity in vivo. Here we introduce volumetric Two-photon Imaging of Neurons using Stereoscopy (vTwINS), a volumetric calcium imaging method that employs an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced “image pairs” in the resulting 2D image, and the separation distance between images is proportional to depth in the volume. To demix the fluorescence time series of individual neurons, we introduce a novel orthogonal matching pursuit algorithm that also infers source locations within the 3D volume. We illustrate vTwINS by imaging neural population activity in mouse primary visual cortex and hippocampus. Our results demonstrate that vTwINS provides an effective method for volumetric two-photon calcium imaging that increases the number of neurons recorded while maintaining a high frame-rate

Rab proteins not associated with virus particle exocytosis.

<p>Cells were transduced to express mCherry-tagged Rab proteins, infected with PRV 486 expressing gM-pHluorin, and imaged at 4.5–5 hr after PRV infection. (A,D,G,J) The indicated Rab proteins are not present at gM-pHluorin exocytosis event (green circle). Images correspond to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat.1004535.s005" target="_blank">Movie S5</a>. Scale bar represents 2 µm. (B,E,H,K) Kymographs of indicated Rab protein (red) and gM-pHluorin (green) fluorescence over time. (C,F,I,L) Ensemble averages of gM-pHluorin (top, green line) and indicated Rab protein (bottom, red line) relative fluorescence. Shaded area represents standard deviation. (A–C) mCherry-Rab3a. Data represent 38 exocytosis events in 2 independent experiments. (D–F) mCherry-Rab27a. Data represent 23 exocytosis events in 2 independent experiments. (G–I) mCherry-Rab5a. Data represent 37 exocytosis events in 2 independent experiments. (J–L) mCherry-Rab7a. Data represent 30 exocytosis events in 2 independent experiments.</p

Viral exocytosis occurs most frequently near patches of LL5β.

<p>(A–B) Cells were transduced to express mRFP-LL5β, infected with PRV 486 expressing gM-pHluorin, and imaged at 4.5–5 hr after PRV infection. Data represent 150 exocytosis events in 9 independent experiments. Scale bars represent 2 µm in all images. (A) Image is a maximum difference projection depicting exocytosis events over a 10 min time course. Particle exocytosis events are classified according to their proximity to mRFP-LL5β patches (yellow circles), or lack thereof (green squares). (B) Still images of a single exocytosis event, corresponding to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#ppat.1004535.s006" target="_blank">Movie S6</a>. (C) Schematic of molecular and cellular mechanisms that coordinate viral transport and exocytosis. Please refer to the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004535#s3" target="_blank">discussion</a> section for references supporting the depicted molecular links.</p