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Encéfalo , Neurociências , Humanos , Neurociências/métodos , Encéfalo/fisiologia , Modelos Neurológicos , AnimaisRESUMO
Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine drives fast excitation and delayed inhibition of behavior and circuit activity. We find that the inhibitory arm of this feedforward motif is implemented by astroglial purinergic signaling. Neuromodulator imaging, behavioral pharmacology, and perturbations of neurons and astroglia reveal that norepinephrine triggers astroglial release of adenosine triphosphate, extracellular conversion into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. This work, along with a companion piece by Lefton and colleagues demonstrating an analogous pathway mediating the effect of norepinephrine on synaptic connectivity in mice, identifies a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in norepinephrine-mediated behavioral and brain state transitions.
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Astrocytes are predominant glial cells that tile the central nervous system and participate in well-established functional and morphological interactions with neurons, blood vessels, and other glia. These ubiquitous cells display rich intracellular Ca2+ signaling, which has now been studied for over 30 years. In this review, we provide a summary and perspective of recent progress concerning the study of astrocyte intracellular Ca2+ signaling as well as discussion of its potential functions. Progress has occurred in the areas of imaging, silencing, activating, and analyzing astrocyte Ca2+ signals. These insights have collectively permitted exploration of the relationships of astrocyte Ca2+ signals to neural circuit function and behavior in a variety of species. We summarize these aspects along with a framework for mechanistically interpreting behavioral studies to identify directly causal effects. We finish by providing a perspective on new avenues of research concerning astrocyte Ca2+ signaling.
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Optical recording of intricate molecular dynamics is becoming an indispensable technique for biological studies, accelerated by the development of new or improved biosensors and microscopy technology. This creates major computational challenges to extract and quantify biologically meaningful spatiotemporal patterns embedded within complex and rich data sources, many of which cannot be captured with existing methods. Here, we introduce Activity Quantification and Analysis (AQuA2), a fast, accurate, and versatile data analysis platform built upon advanced machine learning techniques. It decomposes complex live imaging-based datasets into elementary signaling events, allowing accurate and unbiased quantification of molecular activities and identification of consensus functional units. We demonstrate applications across a wide range of biosensors, cell types, organs, animal models, and imaging modalities. As exemplar findings, we show how AQuA2 identified drug-dependent interactions between neurons and astroglia, and distinct sensorimotor signal propagation patterns in the mouse spinal cord.
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All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.
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Calcium imaging has been widely adopted for its ability to record from large neuronal populations. To summarize the time course of neural activity, dimensionality reduction methods, which have been applied extensively to population spiking activity, may be particularly useful. However, it is unclear if the dimensionality reduction methods applied to spiking activity are appropriate for calcium imaging. We thus carried out a systematic study of design choices based on standard dimensionality reduction methods. We also developed a method to perform deconvolution and dimensionality reduction simultaneously (Calcium Imaging Linear Dynamical System, CILDS). CILDS most accurately recovered the single-trial, low-dimensional time courses from simulated calcium imaging data. CILDS also outperformed the other methods on calcium imaging recordings from larval zebrafish and mice. More broadly, this study represents a foundation for summarizing calcium imaging recordings of large neuronal populations using dimensionality reduction in diverse experimental settings.
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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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Sinalização do Cálcio , Cálcio , Calmodulina , Neurônios , Óxido Nítrico Sintase Tipo III , Fragmentos de Peptídeos , Cálcio/análise , Cálcio/metabolismo , Calmodulina/metabolismo , Neurônios/metabolismo , Cinética , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo III/metabolismo , Fatores de Tempo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismoRESUMO
Much of systems neuroscience posits the functional importance of brain activity patterns that lack natural scales of sizes, durations, or frequencies. The field has developed prominent, and sometimes competing, explanations for the nature of this scale-free activity. Here, we reconcile these explanations across species and modalities. First, we link estimates of excitation-inhibition (E-I) balance with time-resolved correlation of distributed brain activity. Second, we develop an unbiased method for sampling time series constrained by this time-resolved correlation. Third, we use this method to show that estimates of E-I balance account for diverse scale-free phenomena without need to attribute additional function or importance to these phenomena. Collectively, our results simplify existing explanations of scale-free brain activity and provide stringent tests on future theories that seek to transcend these explanations.
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Neurociências , Fatores de Tempo , Encéfalo/fisiologiaRESUMO
To track and control self-location, animals integrate their movements through space. Representations of self-location are observed in the mammalian hippocampal formation, but it is unknown if positional representations exist in more ancient brain regions, how they arise from integrated self-motion, and by what pathways they control locomotion. Here, in a head-fixed, fictive-swimming, virtual-reality preparation, we exposed larval zebrafish to a variety of involuntary displacements. They tracked these displacements and, many seconds later, moved toward their earlier location through corrective swimming ("positional homeostasis"). Whole-brain functional imaging revealed a network in the medulla that stores a memory of location and induces an error signal in the inferior olive to drive future corrective swimming. Optogenetically manipulating medullary integrator cells evoked displacement-memory behavior. Ablating them, or downstream olivary neurons, abolished displacement corrections. These results reveal a multiregional hindbrain circuit in vertebrates that integrates self-motion and stores self-location to control locomotor behavior.
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Neurônios , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Neurônios/fisiologia , Rombencéfalo/fisiologia , Encéfalo/fisiologia , Natação/fisiologia , Homeostase , MamíferosRESUMO
Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of large populations of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We characterized a previously undescribed subpopulation of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish.
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Neurônios Motores , Peixe-Zebra , Animais , Locomoção/fisiologia , Neurônios Motores/fisiologia , Medula Espinal/fisiologia , Natação , Peixe-Zebra/fisiologiaRESUMO
The small size and translucency of larval zebrafish (Danio rerio) have made it a unique experimental system to investigate whole-brain neural circuit structure and function. Still, the connectivity patterns between most neuronal types remain mostly unknown. This gap in knowledge underscores the critical need for effective neural circuit mapping tools, especially ones that can integrate structural and functional analyses. To address this, we previously developed a vesicular stomatitis virus (VSV) based approach called Tracer with Restricted Anterograde Spread (TRAS). TRAS utilizes lentivirus to complement replication-incompetent VSV (VSVΔG) to allow restricted (monosynaptic) anterograde labeling from projection neurons to their target cells in the brain. Here, we report the second generation of TRAS (TRAS-M51R), which utilizes a mutant variant of VSVΔG [VSV(M51R)ΔG] with reduced cytotoxicity. Within the primary visual pathway, we found that TRAS-M51R significantly improved long-term viability of transsynaptic labeling (compared to TRAS) while maintaining anterograde spread activity. By using Cre-expressing VSV(M51R)ΔG, TRAS-M51R could selectively label excitatory (vglut2a positive) and inhibitory (gad1b positive) retinorecipient neurons. We further show that these labeled excitatory and inhibitory retinorecipient neurons retained neuronal excitability upon visual stimulation at 5-8 days post fertilization (2-5 days post-infection). Together, these findings show that TRAS-M51R is suitable for neural circuit studies that integrate structural connectivity, cell-type identity, and neurophysiology.
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The brain is tasked with choosing actions that maximize an animal's chances of survival and reproduction. These choices must be flexible and informed by the current state of the environment, the needs of the body, and the outcomes of past actions. This information is physiologically encoded and processed across different brain regions on a wide range of spatial scales, from molecules in single synapses to networks of brain areas. Uncovering these spatially distributed neural interactions underlying behavior requires investigations that span a similar range of spatial scales. Larval zebrafish, given their small size, transparency, and ease of genetic access, are a good model organism for such investigations, allowing the use of modern microscopy, molecular biology, and computational techniques. These approaches are yielding new insights into the mechanistic basis of behavioral states, which we review here and compare to related studies in mammalian species.
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Fenômenos Fisiológicos do Sistema Nervoso , Peixe-Zebra , Animais , Encéfalo , Larva , SinapsesRESUMO
Genetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost 2 decades of steady improvements in the Aequorea victoria GFP-based GCaMP series of GECIs, the performance of the most recent generation (i.e., jGCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression toward ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.
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Cálcio , Neurônios , Peixe-Zebra , Animais , Linhagem Celular , Células Cultivadas , Peixe-Zebra/genéticaRESUMO
Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 µm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics.
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Encéfalo/diagnóstico por imagem , Cálcio/metabolismo , Corpo Celular/patologia , Neurônios/patologia , Imagem Óptica/métodos , Animais , Artefatos , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio , Corpo Celular/metabolismo , Proteínas de Fluorescência Verde , Camundongos , Neurônios/metabolismo , Neurópilo , Peixe-ZebraRESUMO
Brains are notoriously hard to understand, and neuroscientists need all the tools they can get their hands on to have a realistic shot at it. Advances in machine learning are proving instrumental, illustrated by their recent use to shed light on navigational strategies implemented by zebrafish brains.
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Neurociências , Peixe-Zebra , Animais , Encéfalo , Redes Neurais de Computação , TemperaturaRESUMO
Medial and lateral hypothalamic loci are known to suppress and enhance appetite, respectively, but the dynamics and functional significance of their interaction have yet to be explored. Here we report that, in larval zebrafish, primarily serotonergic neurons of the ventromedial caudal hypothalamus (cH) become increasingly active during food deprivation, whereas activity in the lateral hypothalamus (LH) is reduced. Exposure to food sensory and consummatory cues reverses the activity patterns of these two nuclei, consistent with their representation of opposing internal hunger states. Baseline activity is restored as food-deprived animals return to satiety via voracious feeding. The antagonistic relationship and functional importance of cH and LH activity patterns were confirmed by targeted stimulation and ablation of cH neurons. Collectively, the data allow us to propose a model in which these hypothalamic nuclei regulate different phases of hunger and satiety and coordinate energy balance via antagonistic control of distinct behavioral outputs.
How soon after a meal do you start feeling hungry again? The answer depends on a complex set of processes within the brain that regulate appetite. A key player in these processes is the hypothalamus, a small structure at the base of the brain. The hypothalamus consists of many different subregions, some of which are responsible for increasing or decreasing hunger. Wee, Song et al. now show how two of these subregions interact to regulate appetite and feeding, by studying them in hungry zebrafish larvae. The brains of zebrafish have many features in common with the brains of mammals, but they are smaller and transparent, which makes them easier to study. Wee, Song et al. show that as larvae become hungry, an area called the caudal hypothalamus increases its activity. But when the larvae find food and start feeding, activity in this area falls sharply. It then remains low while the hungry larvae eat as much as possible. Eventually the larvae become full and start eating more slowly. As they do so, the activity of the caudal hypothalamus goes back to normal levels. While this is happening, activity in a different area called the lateral hypothalamus shows the opposite pattern. It has low activity in hungry larvae, which increases when food becomes available and feeding begins. When the larvae finally reduce their rate of feeding, the activity in the lateral hypothalamus drops back down. The authors posit that by inhibiting each other's activity, the caudal and lateral hypothalamus work together to ensure that animals search for food when necessary, but switch to feeding behavior when food becomes available. Serotonin which is produced by the caudal hypothalamus and drugs that act like it have been proposed to suppress appetite, but they have varied and complex effects on food intake and weight gain. By showing that activity in the caudal hypothalamus changes depending on whether food is present, the current findings may provide insights into this complexity. More generally, they show that mapping the circuits that regulate appetite and feeding in simple organisms could help us understand the same processes in humans.
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Apetite , Hipotálamo/fisiologia , Rede Nervosa/fisiologia , Neurônios Serotoninérgicos/fisiologia , Peixe-Zebra/fisiologia , Animais , Larva/fisiologiaRESUMO
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.
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Monitorização Fisiológica/métodos , Neuroimagem/métodos , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Comportamento Animal , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Engenharia Genética , Larva , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mesencéfalo/citologia , Mesencéfalo/fisiologia , Camundongos , Optogenética , Domínios Proteicos , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Natação , Peixe-ZebraRESUMO
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 Biossensoriais/métodos , Encéfalo/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Imagem Molecular/métodos , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Anestesia , Animais , Animais Geneticamente Modificados , Feminino , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Convulsões/metabolismo , Convulsões/patologia , Peixe-ZebraRESUMO
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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Astrócitos/metabolismo , Comportamento Animal/fisiologia , Larva/fisiologia , Peixe-Zebra/fisiologia , Neurônios Adrenérgicos/metabolismo , Animais , Animais Geneticamente Modificados/fisiologia , Astrócitos/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/fisiologia , Mapeamento Encefálico , Cálcio/metabolismo , Comunicação Celular/fisiologia , Retroalimentação Sensorial/fisiologia , Neurônios GABAérgicos/metabolismo , Potenciais da Membrana/fisiologia , Optogenética , Natação/fisiologiaRESUMO
Whole-brain imaging allows for comprehensive functional mapping of distributed neural pathways, but neuronal perturbation experiments are usually limited to targeting predefined regions or genetically identifiable cell types. To complement whole-brain measures of activity with brain-wide manipulations for testing causal interactions, we introduce a system that uses measured activity patterns to guide optical perturbations of any subset of neurons in the same fictively behaving larval zebrafish. First, a light-sheet microscope collects whole-brain data that are rapidly analyzed by a distributed computing system to generate functional brain maps. On the basis of these maps, the experimenter can then optically ablate neurons and image activity changes across the brain. We applied this method to characterize contributions of behaviorally tuned populations to the optomotor response. We extended the system to optogenetically stimulate arbitrary subsets of neurons during whole-brain imaging. These open-source methods enable delineating the contributions of neurons to brain-wide circuit dynamics and behavior in individual animals.