UNLABELLED: Temporal limits on perceptual decisions set strict boundaries on the possible underlying neural computations. How odor information is encoded in the olfactory system is still poorly understood. Here, we sought to define the limit on the speed of olfactory processing. To achieve this, we trained mice to discriminate different odor concentrations in a novel behavioral setup with precise odor delivery synchronized to the sniffing cycle. Mice reported their choice by moving a horizontal treadmill with their front limbs. We found that mice reported discriminations of 75% accuracy in 70-90 ms after odor inhalation. For a low concentration and nontrigeminal odorant, this time was 90-140 ms, showing that mice process odor information rapidly even in the absence of trigeminal stimulation. These response times establish, after accounting for odor transduction and motor delays, that olfactory processing can take tens of milliseconds. This study puts a strong limit on the underlying neural computations and suggests that the action potentials forming the neural basis for these decisions are fired in a few tens of milliseconds.

SIGNIFICANCE STATEMENT: Understanding how sensory information is processed requires different approaches that span multiple levels of investigation from genes to neurons to behavior. Limits on behavioral performance constrain the possible neural mechanisms responsible for specific computations. Using a novel behavioral paradigm, we established that mice can make decisions about odor intensity surprisingly fast. After accounting for sensory and motor delays, the limit on some olfactory neural computations can be as low as a few tens of milliseconds, which suggests that only the first action potentials across a population of neurons contribute to these computations.

Development of cell-based odorant sensor elements combined not only high degree of sensitivity and selectivity but also long-term stability is crucial for their practical applications. Here we report the development of a novel cell-based odorant sensor element that sensitively and selectively detects odorants and displays increased fluorescent intensities over a long period of time. Our odorant sensor elements, based on Sf21 cell lines expressing insect odorant receptors, are sensitive to the level of several tens of parts per billion in solution, can selectively distinguish between different types of odorants based on the odorant selectivity intrinsic to the expressed receptors, and have response times of approximately 13s. Specifically, with the use of Sf21 cells and insect odorant receptors, we demonstrated that the established cell lines stably expressing insect odorant receptors are able to detect odorants with consistent responsiveness for at least 2 months, thus exceeding the short life-span normally associated with cell-based sensors. We also demonstrated the development of a compact odorant sensor chip by integrating the established insect cell lines into a microfluidic chip. The methodology we established in this study, in conjunction with the large repertoire of insect odorant receptors, will aid in the development of practical cell-based odorant sensors for various applications, including food administration and health management.

Mitochondrial dysfunction has been associated with schizophrenia (SZ) and bipolar disorder (BD). This review examines recent publications and novel associations between mitochondrial genes and SZ and BD. Associations of nuclear-encoded mitochondrial variants with SZ were found using gene- and pathway-based approaches. Two control region mitochondrial DNA (mtDNA) SNPs, T16519C and T195C, both showed an association with SZ and BD. A review of 4 studies of A15218G located in the cytochrome B oxidase gene (CYTB, SZ = 11,311, control = 35,735) shows a moderate association with SZ ( = 2.15E-03). Another mtDNA allele A12308G was nominally associated with psychosis in BD type I subjects and SZ. The first published study testing the epistatic interaction between nuclear-encoded and mitochondria-encoded genes demonstrated evidence for potential interactions between mtDNA and the nuclear genome for BD. A similar analysis for the risk of SZ revealed significant joint effects (34 nuclear-mitochondria SNP pairs with joint effect ≤ 5E-07) and significant enrichment of projection neurons. The mitochondria-encoded gene CYTB was found in both the epistatic interactions for SZ and BD and the single SNP association of SZ. Future efforts considering population stratification and polygenic risk scores will test the role of mitochondrial variants in psychiatric disorders.

The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [3H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF549-conjugated DG3-80 (3) and JF646-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

An important question in early neural development is the origin of stochastic nuclear movement between apical and basal surfaces of neuroepithelia during interkinetic nuclear migration. Tracking of nuclear subpopulations has shown evidence of diffusion - mean squared displacements growing linearly in time - and suggested crowding from cell division at the apical surface drives basalward motion. Yet, this hypothesis has not yet been tested, and the forces involved not quantified. We employ long-term, rapid light-sheet and two-photon imaging of early zebrafish retinogenesis to track entire populations of nuclei within the tissue. The time-varying concentration profiles show clear evidence of crowding as nuclei reach close-packing and are quantitatively described by a nonlinear diffusion model. Considerations of nuclear motion constrained inside the enveloping cell membrane show that concentration-dependent stochastic forces inside cells, compatible in magnitude to those found in cytoskeletal transport, can explain the observed magnitude of the diffusion constant.

SignificanceInteractions between the cell nucleus and cytoskeleton regulate cell mechanics and are facilitated by the interplay between the nuclear lamina and linker of nucleoskeleton and cytoskeleton (LINC) complexes. To date, the specific contribution of the four lamin isoforms to nucleocytoskeletal connectivity and whole-cell mechanics remains unknown. We discover that A- and B-type lamins distinctively interact with LINC complexes that bind F-actin and vimentin filaments to differentially modulate cortical stiffness, cytoplasmic stiffness, and contractility of mouse embryonic fibroblasts (MEFs). We propose and experimentally verify an integrated lamin-LINC complex-cytoskeleton model that explains cellular mechanical phenotypes in lamin-deficient MEFs. Our findings uncover potential mechanisms for cellular defects in human laminopathies and many cancers associated with mutations or modifications in lamin isoforms.

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogastershavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.

Nuclear receptors (NRs) comprise a family of ligand-regulated transcription factors that control diverse critical biological processes including various aspects of brain development. Eighteen NR genes exist in the Drosophila genome. To explore their roles in brain development, we knocked down individual NRs through the development of the mushroom bodies (MBs) by targeted RNAi. Besides recapitulating the known MB phenotypes for three NRs, we found that unfulfilled (unf), an ortholog of human photoreceptor specific nuclear receptor (PNR), regulates axonal morphogenesis and neuronal subtype identity. The adult MBs develop through remodeling of gamma neurons plus de-novo elaboration of both alpha’/beta’ and alpha/beta neurons. Notably, unf is largely dispensable for the initial elaboration of gamma neurons, but plays an essential role in their re-extension of axons after pruning during early metamorphosis. The subsequently derived MB neuron types also require unf for extension of axons beyond the terminus of the pruned bundle. Tracing single axons revealed misrouting rather than simple truncation. Further, silencing unf in single-cell clones elicited misguidance of axons in otherwise unperturbed MBs. Such axon guidance defects may occur as MB neurons partially lose their subtype identity, as evidenced by suppression of various MB subtype markers in unf knockdown MBs. In sum, unf governs axonal morphogenesis of multiple MB neuron types, possibly through regulating neuronal subtype identity.

The insect mushroom body (MB) is a conserved brain structure that plays key roles in a diverse array of behaviors. The MB is the primary invertebrate model of neural circuits related to memory formation and storage, and its development, morphology, wiring, and function has been extensively studied. MBs consist of intrinsic Kenyon Cells that are divided into three major neuron classes (γ, α'/β' and α/β) and 7 cell subtypes (γd, γm, α'/β'ap, α'/β'm, α/βp, α/βs and α/βc) based on their birth order, morphology, and connectivity. These subtypes play distinct roles in memory processing, however the underlying transcriptional differences are unknown. Here, we used RNA sequencing (RNA-seq) to profile the nuclear transcriptomes of each MB neuronal cell subtypes. We identified 350 MB class- or subtype-specific genes, including the widely used α/β class marker and the α'/β' class marker Immunostaining corroborates the RNA-seq measurements at the protein level for several cases. Importantly, our data provide a full accounting of the neurotransmitter receptors, transporters, neurotransmitter biosynthetic enzymes, neuropeptides, and neuropeptide receptors expressed within each of these cell types. This high-quality, cell type-level transcriptome catalog for the MB provides a valuable resource for the fly neuroscience community.