Major resources are now available to develop tools and technologies aimed at dissecting the circuitry and computations underlying behavior, unraveling the underpinnings of brain disorders, and understanding the neural substrates of cognition. Scientists from around the world shared their views around new tools and technologies to drive advances in neuroscience.

Neural stem cells show age-dependent developmental potentials, as evidenced by their production of distinct neuron types at different developmental times. Drosophila neuroblasts produce long, stereotyped lineages of neurons. We searched for factors that could regulate neural temporal fate by RNA-sequencing lineage-specific neuroblasts at various developmental times. We found that two RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), display opposing high-to-low and low-to-high temporal gradients with lineage-specific temporal dynamics. Imp and Syp promote early and late fates, respectively, in both a slowly progressing and a rapidly changing lineage. Imp and Syp control neuronal fates in the mushroom body lineages by regulating the temporal transcription factor Chinmo translation. Together, the opposing Imp/Syp gradients encode stem cell age, specifying multiple cell fates within a lineage.

Two-photon excitation fluorescence microscopy has revolutionized our understanding of brain structure and function through the high resolution and large penetration depth it offers. Investigating neural structures in vivo requires gaining optical access to the brain, which is typically achieved by replacing a part of the skull with one or several layers of cover glass windows. To compensate for the spherical aberrations caused by the presence of these layers of glass, collar-correction objectives are typically used. However, the efficiency of this correction has been shown to depend significantly on the tilt angle between the glass window surface and the optical axis of the imaging system. Here, we first expand these observations and characterize the effect of the tilt angle on the collected fluorescence signal with thicker windows (double cover slide) and compare these results with an objective devoid of collar-correction. Second, we present a simple optical alignment device designed to rapidly minimize the tilt angle in vivo and align the optical axis of the microscope perpendicularly to the glass window to an angle below 0.25°, thereby significantly improving the imaging quality. Finally, we describe a tilt-correction procedure for users in an in vivo setting, enabling the accurate alignment with a resolution of <0.2° in only few iterations.

Receptor tyrosine kinases (RTK) are important cell surface receptors that transduce extracellular signals across the plasma membrane. The traditional view of how these receptors function is that ligand binding to the extracellular domains acts as a master-switch that enables receptor monomers to dimerize and subsequently trans-phosphorylate each other on their intracellular domains. However, a growing body of evidence suggests that receptor oligomerization is not merely a consequence of ligand binding, but is instead part of a complex process responsible for regulation of receptor activation. Importantly, the oligomerization dynamics and subsequent activation of these receptors are affected by other cellular components, such as cytoskeletal machineries and cell membrane lipid characteristics. Thus receptor activation is not an isolated molecular event mediated by the ligand-receptor interaction, but instead involves orchestrated interactions between the receptors and other cellular components. Measuring receptor oligomerization dynamics on live cells can yield important insights into the characteristics of these interactions. Therefore, it is imperative to develop techniques that can probe receptor movements on the plasma membrane with optimal temporal and spatial resolutions. Various microscopic techniques have been used for this purpose. Optical techniques including single molecule tracking (SMT) and fluorescence correlation spectroscopy (FCS) measure receptor diffusion on live cells. Receptor-receptor interactions can also be assessed by detecting Förster resonance energy transfer (FRET) between fluorescently-labeled receptors situated in close proximity or by counting the number of receptors within a diffraction limited fluorescence spot (stepwise bleaching). This review will describe recent developments of optical techniques that have been used to study receptor oligomerization on living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Sighted animals use visual signals to discern directional motion in their environment. Motion is not directly detected by visual neurons, and it must instead be computed from light signals that vary over space and time. This makes visual motion estimation a near universal neural computation, and decades of research have revealed much about the algorithms and mechanisms that generate directional signals. The idea that sensory systems are optimized for performance in natural environments has deeply impacted this research. In this article, we review the many ways that optimization has been used to quantitatively model visual motion estimation and reveal its underlying principles. We emphasize that no single optimization theory has dominated the literature. Instead, researchers have adeptly incorporated different computational demands and biological constraints that are pertinent to the specific brain system and animal model under study. The successes and failures of the resulting optimization models have thereby provided insights into how computational demands and biological constraints together shape neural computation.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo . Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3.GCaMP5allows more sensitive detection of neural activity in vivo andmayfind widespread applications for cellular imaging in general.

Correlative light and electron microscopy (CLEM) combines the power of electron microscopy, with its excellent resolution and contrast, with that of fluorescence imaging, which allows the staining of specific molecules, organelles, and cell populations. Fluorescence imaging is also readily compatible with live cells and behaving animals, facilitating real-time visualization of cellular processes, potentially followed by electron microscopic reconstruction. Super-resolution single-molecule localization microscopy is a relatively new modality that harnesses the ability of some fluorophores to photoconvert, through which localization precision better than Abbe’s diffraction limit is achieved through iterative high-resolution localization of single-molecule emitters. Here we describe our lab’s recent progress in the development of reagents and techniques for super-resolution single-molecule localization CLEM and their applications to biological problems.

The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These ’Twitch’ sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.