Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

Both neurons and glia communicate through diffusible neuromodulators; however, how neuron-glial interactions in such neuromodulatory networks influence circuit computation and behavior is unclear. During futility-induced behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine (NE) drives fast excitation and delayed inhibition of behavior and circuit activity. We found that astroglial purinergic signaling implements the inhibitory arm of this motif. In larval zebrafish, NE triggers astroglial release of adenosine triphosphate (ATP), extracellular conversion of ATP into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. Our results suggest a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in NE-mediated behavioral and brain state transitions and position astroglia as important effectors in neuromodulatory signaling.

Preprint: https://www.biorxiv.org/content/early/2024/05/23/2024.05.23.595576

Sensory areas are spontaneously active in the absence of sensory stimuli. This spontaneous activity has long been studied; however, its functional role remains largely unknown. Recent advances in technology, allowing large-scale neural recordings in the awake and behaving animal, have transformed our understanding of spontaneous activity. Studies using these recordings have discovered high-dimensional spontaneous activity patterns, correlation between spontaneous activity and behavior, and dissimilarity between spontaneous and sensory-driven activity patterns. These findings are supported by evidence from developing animals, where a transition toward these characteristics is observed as the circuit matures, as well as by evidence from mature animals across species. These newly revealed characteristics call for the formulation of a new role for spontaneous activity in neural sensory computation.

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.

NPAS4 is an activity-dependent transcription factor that, in CA1 of the hippocampus, regulates inhibitory synapses made onto the active pyramidal neuron. In principle, NPAS4 thereby allows the past activity of a neuron to influence how it encodes information, although this has not yet been demonstrated. Here, we generated a sparse, CA1-specific knockout (KO) of NPAS4 in the mouse hippocampus and used optogenetic tagging to identify KO neurons in vivo. Recordings from intermingled wild-type (WT) and KO neurons in awake behaving animals revealed that NPAS4 deletion degrades spatial representations and temporal precision of spiking: KO neurons exhibited larger place fields with reduced in-field firing and increased out-of-field firing, less stable place fields, reduced coupling to local field potential theta oscillations, and diminished phase precession. These findings demonstrate that NPAS4 plays a crucial role in refining the spatial and temporal properties of CA1 pyramidal neuron spikes, which themselves are thought to be fundamental building blocks of more complex processes such as learning and memory.

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.