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Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.
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Evolución Molecular Dirigida , Aprendizaje Automático , Serotonina/metabolismo , Algoritmos , Secuencia de Aminoácidos , Amígdala del Cerebelo/fisiología , Animales , Conducta Animal , Sitios de Unión , Encéfalo/metabolismo , Células HEK293 , Humanos , Cinética , Modelos Lineales , Ratones , Ratones Endogámicos C57BL , Fotones , Unión Proteica , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sueño/fisiología , Vigilia/fisiologíaRESUMEN
Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis. VIDEO ABSTRACT.
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When a behavior repeatedly fails to achieve its goal, animals often give up and become passive, which can be strategic for preserving energy or regrouping between attempts. It is unknown how the brain identifies behavioral failures and mediates this behavioral-state switch. In larval zebrafish swimming in virtual reality, visual feedback can be withheld so that swim attempts fail to trigger expected visual flow. After tens of seconds of such motor futility, animals became passive for similar durations. Whole-brain calcium imaging revealed noradrenergic neurons that responded specifically to failed swim attempts and radial astrocytes whose calcium levels accumulated with increasing numbers of failed attempts. Using cell ablation and optogenetic or chemogenetic activation, we found that noradrenergic neurons progressively activated brainstem radial astrocytes, which then suppressed swimming. Thus, radial astrocytes perform a computation critical for behavior: they accumulate evidence that current actions are ineffective and consequently drive changes in behavioral states. VIDEO ABSTRACT.
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Astrocitos/metabolismo , Conducta Animal/fisiología , Larva/fisiología , Pez Cebra/fisiología , Neuronas Adrenérgicas/metabolismo , Animales , Animales Modificados Genéticamente/fisiología , Astrocitos/citología , Encéfalo/diagnóstico por imagen , Encéfalo/fisiología , Mapeo Encefálico , Calcio/metabolismo , Comunicación Celular/fisiología , Retroalimentación Sensorial/fisiología , Neuronas GABAérgicas/metabolismo , Potenciales de la Membrana/fisiología , Optogenética , Natación/fisiologíaRESUMEN
Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.
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Señalización del Calcio , Calcio , Calmodulina , Neuronas , Óxido Nítrico Sintasa de Tipo III , Fragmentos de Péptidos , Calcio/análisis , Calcio/metabolismo , Calmodulina/metabolismo , Neuronas/metabolismo , Cinética , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Factores de Tiempo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismoRESUMEN
Chemotactic bacteria not only navigate chemical gradients, but also shape their environments by consuming and secreting attractants. Investigating how these processes influence the dynamics of bacterial populations has been challenging because of a lack of experimental methods for measuring spatial profiles of chemoattractants in real time. Here, we use a fluorescent sensor for aspartate to directly measure bacterially generated chemoattractant gradients during collective migration. Our measurements show that the standard Patlak-Keller-Segel model for collective chemotactic bacterial migration breaks down at high cell densities. To address this, we propose modifications to the model that consider the impact of cell density on bacterial chemotaxis and attractant consumption. With these changes, the model explains our experimental data across all cell densities, offering insight into chemotactic dynamics. Our findings highlight the significance of considering cell density effects on bacterial behavior, and the potential for fluorescent metabolite sensors to shed light on the complex emergent dynamics of bacterial communities.
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Factores Quimiotácticos , Quimiotaxis , Transporte Biológico , Ácido Aspártico , ColorantesRESUMEN
The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.
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Ácido Glutámico , Transmisión Sináptica , Ratones , Animales , Ácido Glutámico/metabolismo , Cinética , Neuronas/fisiología , Sinapsis/fisiologíaRESUMEN
We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.
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Transferencia Resonante de Energía de Fluorescencia , Microscopía , Proteínas Luminiscentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , LuzRESUMEN
Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. We used fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes were accompanied by brief glutamate transients which were maximal at the initiation site and rapidly propagated centrifugally. GABA transients lasted longer than glutamate transients and were maximal â¼1.5â mm from the focus where they propagated centripetally. Prior to seizure initiation GABA transients were attenuated, whilst glutamate transients increased, consistent with a progressive failure of local inhibitory restraint. As seizures increased in frequency, there was a gradual increase in the spatial extent of spike-associated glutamate transients associated with interictal spikes. Neurotransmitter imaging thus reveals a progressive collapse of an annulus of feed-forward GABA release, allowing seizures to escape from local inhibitory restraint.
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Epilepsias Parciales , Ácido Glutámico , Humanos , Convulsiones , Cognición , Ácido gamma-AminobutíricoRESUMEN
We review the principles of development and deployment of genetically encoded calcium indicators (GECIs) for the detection of neural activity. Our focus is on the popular GCaMP family of green GECIs, culminating in the recent release of the jGCaMP8 sensors, with dramatically improved kinetics relative to previous generations. We summarize the properties of GECIs in multiple colour channels (blue, cyan, green, yellow, red, far-red) and highlight areas for further improvement. With their low-millisecond rise-times, the jGCaMP8 indicators allow new classes of experiments following neural activity in time frames approaching the underlying computations.
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OBJECTIVE: A recent genome-wide association study linked KLF2 as a novel Asian-specific locus for systemic lupus erythematosus (SLE) susceptibility. However, the underlying causal functional variant(s), cognate target gene(s) and genetic mechanisms associated with SLE risk are unknown. METHODS: We used bioinformatics to prioritise likely functional variants and validated the best candidate with diverse experimental techniques, including genome editing. Gene expression was compared between healthy controls (HCs) and patients with SLE with or without lupus nephritis (LN+, LN-). RESULTS: Through bioinformatics and expression quantitative trait locus analyses, we prioritised rs4808485 in active chromatin, predicted to modulate KLF2 expression. Luciferase reporter assays and chromatin immunoprecipitation-qPCR demonstrated differential allele-specific enhancer activity and binding of active histone marks (H3K27ac, H3K4me3 and H3K4me1), Pol II, CTCF, P300 and the transcription factor PARP1. Chromosome conformation capture-qPCR revealed long-range chromatin interactions between rs4808485 and the KLF2 promoter. These were directly validated by CRISPR-based genetic and epigenetic editing in Jurkat and lymphoblastoid cells. Deleting the rs4808485 enhancer in Jurkat (KO) cells disrupted NLRP3 inflammasome machinery by reducing KLF2 and increasing CASPASE1, IL-1ß and GSDMD levels. Knockout cells also exhibited higher proliferation and cell-cycle progression than wild type. RNA-seq validated interplay between KLF2 and inflammasome machinery in HC, LN+ and LN-. CONCLUSIONS: We demonstrate how rs4808485 modulates the inflammasome and cellular homoeostasis through regulating KLF2 expression. This establishes mechanistic connections between rs4808485 and SLE susceptibility.
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Predisposición Genética a la Enfermedad , Homeostasis , Inflamasomas , Factores de Transcripción de Tipo Kruppel , Lupus Eritematoso Sistémico , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Inflamasomas/genética , Lupus Eritematoso Sistémico/genética , Homeostasis/genética , Polimorfismo de Nucleótido Simple , Estudio de Asociación del Genoma Completo , Sitios de Carácter Cuantitativo , Nefritis Lúpica/genética , Estudios de Casos y Controles , Elementos de Facilitación Genéticos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.
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Vías Eferentes/citología , Vías Eferentes/fisiología , Corteza Motora/citología , Corteza Motora/fisiología , Movimiento/fisiología , Animales , Ganglios Basales/citología , Tronco Encefálico/citología , Ácido Glutámico/metabolismo , Bulbo Raquídeo/citología , Ratones , Neuronas/metabolismo , Células Piramidales/clasificación , Células Piramidales/fisiología , Análisis de la Célula Individual , TranscriptomaRESUMEN
Transmembrane transporter proteins allow the passage of essentially all biologically important molecules across the lipid membranes of cells and organelles and are therefore of central importance to all forms of life. Current methods of transporter measurement, however, are lacking in several dimensions. Herein, a method is presented in which oscillating stimuli are presented to transporter-expressing cells, and activity is measured through imaging the corresponding oscillating responses of intracellular fluorescent sensors. This approach yields continuous temporal readouts of transporter activity and can therefore be used to measure time-dependent responses to drugs and other stimuli. Because of the periodic nature of the response, temporal Fourier transforms can be used to identify and quantify regions of interest in the xy plane and to overcome noise. This technique, called the Oscillating Stimulus Transporter Assay (OSTA), should greatly facilitate both functional characterization of transporters as well as high-throughput screening of drugs for transporters of particular pathophysiological interest.
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Benzopiranos/química , Bioensayo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Colorantes Fluorescentes/química , Imagen Óptica/métodos , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Compuestos de Bencidrilo/farmacología , Transporte Biológico , Membrana Celular/efectos de los fármacos , Antiportadores de Cloruro-Bicarbonato/genética , Antiportadores de Cloruro-Bicarbonato/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Transportador 2 de Aminoácidos Excitadores , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Glucósidos/farmacología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Células HEK293 , Humanos , Plásmidos/química , Plásmidos/metabolismo , Ácido Salicílico/farmacología , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Transportador 2 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/metabolismo , Transportadores de Sulfato , TransfecciónRESUMEN
Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.
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Calcio/análisis , Colorantes Fluorescentes/química , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Drosophila , Femenino , Rayos Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Imagen Molecular , Corteza Somatosensorial/químicaRESUMEN
We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBP's second shell and hinge regions. iS-methadoneSnFR displays the necessary sensitivity, kinetics, and selectivityânotably enantioselectivity against R-methadoneâfor biological applications. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Expression and imaging in mammalian cells demonstrate that S-methadone enters at least two organelles and undergoes acid trapping in the Golgi apparatus, where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.
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Agonistas Nicotínicos , Proteínas de Unión Periplasmáticas , Animales , Mamíferos/metabolismo , Metadona , Ratones , Mutación , Orgánulos/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.
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Corteza Cerebral/fisiología , Ácido Glutámico/metabolismo , Neuronas/fisiología , Tomografía/métodos , Animales , Calcio/metabolismo , Corteza Cerebral/citología , Femenino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Fotones , RatasRESUMEN
Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.
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Calcio/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Drosophila , Femenino , Proteínas Fluorescentes Verdes , Ratones , Unión Neuromuscular/diagnóstico por imagen , Ratas , Corteza Visual/metabolismoRESUMEN
Current techniques for monitoring GABA (γ-aminobutyric acid), the primary inhibitory neurotransmitter in vertebrates, cannot follow transients in intact neural circuits. To develop a GABA sensor, we applied the design principles used to create the fluorescent glutamate receptor iGluSnFR. We used a protein derived from a previously unsequenced Pseudomonas fluorescens strain and performed structure-guided mutagenesis and library screening to obtain intensity-based GABA sensing fluorescence reporter (iGABASnFR) variants. iGABASnFR is genetically encoded, detects GABA release evoked by electric stimulation of afferent fibers in acute brain slices and produces readily detectable fluorescence increases in vivo in mice and zebrafish. We applied iGABASnFR to track mitochondrial GABA content and its modulation by an anticonvulsant, swimming-evoked, GABA-mediated transmission in zebrafish cerebellum, GABA release events during interictal spikes and seizures in awake mice, and found that GABA-mediated tone decreases during isoflurane anesthesia.
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Técnicas Biosensibles/métodos , Encéfalo/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/metabolismo , Imagen Molecular/métodos , Neuronas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Anestesia , Animales , Animales Modificados Genéticamente , Femenino , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Convulsiones/metabolismo , Convulsiones/patología , Pez CebraRESUMEN
The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594-5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.
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In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.