Real-time lineage tracing in flies gets a boost with three techniques to specifically label a progenitor’s daughter cells.

How memories of past events influence behavior is a key question in neuroscience. The major associative learning center in Drosophila, the Mushroom Body (MB), communicates to the rest of the brain through Mushroom Body Output Neurons (MBONs). While 21 MBON cell types have their dendrites confined to small compartments of the MB lobes, analysis of EM connectomes revealed the presence of an additional 14 MBON cell types that are atypical in having dendritic input both within the MB lobes and in adjacent brain regions. Genetic reagents for manipulating atypical MBONs and experimental data on their functions has been lacking. In this report we describe new cell-type-specific GAL4 drivers for many MBONs, including the majority of atypical MBONs. Using these genetic reagents, we conducted optogenetic activation screening to examine their ability to drive behaviors and learning. These reagents provide important new tools for the study of complex behaviors in Drosophila.

Temporal focusing (TF) multiphoton systems constitute a powerful solution for cellular resolution optogenetic stimulation and recording in three-dimensional, scattering tissue. Here, we address two fundamental aspects in the design of such systems: first, we examine the design of TF systems with specific optical sectioning by comparatively analyzing previously published results. Next, we develop a solution for obtaining TF in a flexible three-dimensional pattern of cellmatched focal spots. Our solution employs spatio-temporal focusing (SSTF) in a unique optical system design that can be integrated before essentially any multiphoton imaging or stimulation system.

Chemical signaling between neurons in the brain can be divided into two major categories: fast synaptic transmission and neuromodulation. Fast synaptic transmission, mediated by amino acids such as glutamate and GABA, occurs on millisecond time scales and results in the influx of ions through ligand-gated ion channels on postsynaptic neurons (Figure 1A). Electrophysiological and optical imaging tools, including genetically encoded voltage indicators, have enabled neuroscientists to link cause (neurotransmitter release) and effect (membrane polarization) of synaptic transmission in time and space. Unlike classical neurotransmitters, neuromodulators do not produce immediate electrical effects that excite or inhibit target neurons. Instead, neuromodulators tune the intrinsic or synaptic properties of neurons, most commonly through interaction with G-protein-coupled receptors (GPCRs) (Figure 1B). Neuromodulators can escape the synaptic cleft and diffuse broadly, allowing them to influence the activity of many neurons in a state-dependent manner. Therefore, the spatial component of neuromodulator flux is fundamentally important. However, the temporal and/or spatial limitations of techniques classically used to study neuromodulation, such as microdialysis and fast-scan cyclic voltammetry (FSCV), make it difficult to interpret how neuromodulator release affects the plasticity or function of target neuronal populations on a moment-to-moment basis. Therefore, tools that can detect neuromodulators with high spatiotemporal resolution are critical for understanding their impact on neural computations that control behavior in health and disease.

2D template matching (2DTM) can be used to detect molecules and their assemblies in cellular cryo-EM images with high positional and orientational accuracy. While 2DTM successfully detects spherical targets such as large ribosomal subunits, challenges remain in detecting smaller and more aspherical targets in various environments. In this work, a novel 2DTM metric, referred to as the 2DTM p-value, is developed to extend the 2DTM framework to more complex applications. The 2DTM p-value combines information from two previously used 2DTM metrics, namely the 2DTM signal-to-noise ratio (SNR) and z-score, which are derived from the cross-correlation coefficient between the target and the template. The 2DTM p-value demonstrates robust detection accuracies under various imaging and sample conditions and outperforms the 2DTM SNR and z-score alone. Specifically, the 2DTM p-value improves the detection of aspherical targets such as a modified artificial tubulin patch particle (500 kDa) and a much smaller clathrin monomer (193 kDa) in simulated data. It also accurately recovers mature 60S ribosomes in yeast lamellae samples, even under conditions of increased Gaussian noise. The new metric will enable the detection of a wider variety of targets in both purified and cellular samples through 2DTM.

Visualization of cellular and molecular processes is an indispensable tool for cell biologists, and innovations in microscopy methods unfailingly lead to new biological discoveries. Today, light microscopy (LM) provides ever-higher spatial and temporal resolution and visualization of biological process over enormous ranges. Electron microscopy (EM) is moving into the atomic resolution regime and allowing cellular analyses that are more physiological and sophisticated in scope. Importantly, much is being gained by combining multiple approaches, (e.g., LM and EM) to take advantage of their complementary strengths. The advent of high-throughput microscopies has led to a common need for sophisticated computational methods to quantitatively analyze huge amounts of data and translate images into new biological insights.

Because of its genetic, molecular, and behavioral tractability, Drosophila has emerged as a powerful model system for studying molecular and cellular mechanisms underlying the development and function of nervous systems. The Drosophila nervous system has fewer neurons and exhibits a lower glia:neuron ratio than is seen in vertebrate nervous systems. Despite the simplicity of the Drosophila nervous system, glial organization in flies is as sophisticated as it is in vertebrates. Furthermore, fly glial cells play vital roles in neural development and behavior. In addition, powerful genetic tools are continuously being created to explore cell function in vivo. In taking advantage of these features, the fly nervous system serves as an excellent model system to study general aspects of glial cell development and function in vivo. In this article, we review and discuss advanced genetic tools that are potentially useful for understanding glial cell biology in Drosophila.

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species, and it is especially useful for analyzing haploid eukaryotes. MLST is rigorous, reproducible, and informative, and MLST genotyping has been shown to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. MLST molecular types often correlate with important phenotypes. Conventional MLST involves the extraction of genomic DNA and the amplification by PCR of several conserved, unlinked gene sequences from a sample of isolates of the taxon under investigation. In some cases, as few as three loci are sufficient to yield definitive results. The amplicons are sequenced, aligned, and compared by phylogenetic methods to distinguish statistically significant differences among individuals and clades. Although MLST is simpler, faster, and less expensive than whole genome sequencing, it is more costly and time-consuming than less reliable genotyping methods (e.g. amplified fragment length polymorphisms). Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in single assay. We demonstrate this methodology by genotyping isolates of the well-characterized, human pathogenic yeast Cryptococcus neoformans.

NF-κB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-κB signaling within the nervous system remains to be clearly defined. Here, we show that the NF-κB and IκB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. We then show that mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, we provide evidence that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon our data we speculate that Dorsal, Cactus, and Pelle could function together, locally at the postsynaptic density, to specify GluR levels.

Parallel processing is an organizing principle of many neural circuits. In the retina, parallel neuronal pathways process signals from rod and cone photoreceptors and support vision over a wide range of light levels. Toward this end, rods and cones form triad synapses with dendrites of distinct bipolar cell types, and the axons or dendrites, respectively, of horizontal cells (HCs). The molecular cues that promote the formation of specific neuronal pathways remain largely unknown. Here, we discover that developing and mature HCs express the leucine-rich repeat (LRR)-containing protein netrin-G ligand 2 (NGL-2). NGL-2 localizes selectively to the tips of HC axons, which form reciprocal connections with rods. In mice with null mutations in Ngl-2 (Ngl-2⁻/⁻), many branches of HC axons fail to stratify in the outer plexiform layer (OPL) and invade the outer nuclear layer. In addition, HC axons expand lateral territories and increase coverage of the OPL, but establish fewer synapses with rods. NGL-2 can form transsynaptic adhesion complexes with netrin-G2, which we show to be expressed by photoreceptors. In Ngl-2⁻/⁻ mice, we find specific defects in the assembly of presynaptic ribbons in rods, indicating that reverse signaling of complexes involving NGL-2 regulates presynaptic maturation. The development of HC dendrites and triad synapses of cone photoreceptors proceeds normally in the absence of NGL-2 and in vivo electrophysiology reveals selective defects in rod-mediated signal transmission in Ngl-2⁻/⁻ mice. Thus, our results identify NGL-2 as a central component of pathway-specific development in the outer retina.