Connections between neuronal populations may be genetically hardwired or random. In the insect olfactory system, projection neurons of the antennal lobe connect randomly to Kenyon cells of the mushroom body. Consequently, while the odor responses of the projection neurons are stereotyped across individuals, the responses of the Kenyon cells are variable. Surprisingly, downstream of Kenyon cells, mushroom body output neurons show stereotypy in their responses. We found that the stereotypy is enabled by the convergence of inputs from many Kenyon cells onto an output neuron, and does not require learning. The stereotypy emerges in the total response of the Kenyon cell population using multiple odor-specific features of the projection neuron responses, benefits from the nonlinearity in the transfer function, depends on the convergence:randomness ratio, and is constrained by sparseness. Together, our results reveal the fundamental mechanisms and constraints with which convergence enables stereotypy in sensory responses despite random connectivity.

Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast-KD, B2, B3, and R-are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice.

Glomeruli are functional units in the olfactory system. The mouse olfactory bulb contains roughly 2,000 glomeruli, each receiving inputs from olfactory sensory neurons (OSNs) that express a specific odorant receptor gene. Odors typically activate many glomeruli in complex combinatorial patterns and it is unknown which features of neuronal activity in individual glomeruli contribute to odor perception. To address this, we used optogenetics to selectively activate single, genetically identified glomeruli in behaving mice. We found that mice could perceive the stimulation of a single glomerulus. Single-glomerulus stimulation was also detected on an intense odor background. In addition, different input intensities and the timing of input relative to sniffing were discriminated through one glomerulus. Our data suggest that each glomerulus can transmit odor information using identity, intensity and temporal coding cues. These multiple modes of information transmission may enable the olfactory system to efficiently identify and localize odor sources.

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.

Information processing by brain circuits depends on Ca-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca indicator Cal-590 is Ca-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca or spike-evoked Ca entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.

3-photon excitation enables in vivo fluorescence microscopy deep in densely labeled and highly scattering samples, while maintaining high resolution and contrast. We designed and characterized a dual-plane 3-photon microscope with temporal multiplexing and remote focusing, and performed simultaneous in vivo calcium imaging of two planes deep in the cortex of a transgenic mouse expressing GCaMP6s in nearly all excitatory neurons.

We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein-labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.

We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10 ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kb of its true position using 96 F1-backcross individuals genotyped in a single lane on an Illumina Genome Analyzer. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38-kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.

We consider the problem of optimizing general convex objective functions with nonnegativity constraints. Using the Karush-Kuhn-Tucker (KKT) conditions for the nonnegativity constraints we will derive fast multiplicative update rules for several problems of interest in signal processing, including non-negative deconvolution, point-process smoothing, ML estimation for Poisson Observations, nonnegative least squares and nonnegative matrix factorization (NMF). Our algorithm can also account for temporal and spatial structure and regularization. We will analyze the performance of our algorithm on simultaneously recorded neuronal calcium imaging and electrophysiology data.