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In dynamic environments, animals make behavioral decisions based on the innate valences of sensory cues and information learnt about these cues across multiple timescales1-3. However, it remains unclear how the innate valence of a sensory stimulus affects acquisition of learnt valence information and subsequent memory dynamics. Here we show that in the Drosophila brain, interconnected short- and long-term memory units of the mushroom body jointly regulate memory via dopamine signals that encode innate and learnt sensory valences. Through time-lapse, in vivo voltage-imaging studies of neural spiking in >500 flies undergoing olfactory associative conditioning, we found that protocerebral posterior lateral 1 dopamine neurons (PPL1-DANs)4 heterogeneously and bi-directionally encode innate and learnt valences of punishment, reward, and odor cues. During learning, these valence signals regulate memory storage and extinction in mushroom body output neurons (MBONs)5. In initial conditioning bouts, PPL1-γ1pedc and PPL1-γ2α'1 neurons control short-term memory formation, which weakens inhibitory feedback from MBON-γ1pedc>α/ß to PPL1-α'2α2 and PPL1-α3. During further conditioning, this diminished feedback allows these two PPL1-DANs to encode the net innate plus learnt valence of the conditioned odor cue, which gates long-term memory formation. A computational model constrained by the fly connectome6,7 and our spiking data explains how dopamine signals mediate the circuit interactions between short- and long-term memory traces, yielding predictions that our experiments confirm. Overall, the mushroom body achieves flexible learning via the integration of innate and learnt valences within parallel learning units sharing feedback interconnections. This hybrid physiologic-anatomic mechanism may be a general means by which dopamine regulates memory dynamics in other species and brain structures, including the vertebrate basal ganglia.
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Nervous systems process information by integrating the electrical activity of neurons in complex networks. This motivates the long-standing interest in using optical methods to simultaneously monitor the membrane potential of multiple genetically targeted neurons via expression of genetically encoded fluorescent voltage indicators (GEVIs) in intact neural circuits. No currently available GEVIs have demonstrated robust signals in intact brain tissue that enable reliable recording of individual electrical events simultaneously in multiple neurons. Here, we show that the recently developed "ArcLight" GEVI robustly reports both subthreshold events and action potentials in genetically targeted neurons in the intact Drosophila fruit fly brain and reveals electrical signals in neurite branches. In the same way that genetically encoded fluorescent sensors have revolutionized the study of intracellular Ca(2+) signals, ArcLight now enables optical measurement in intact neural circuits of membrane potential, the key cellular parameter that underlies neuronal information processing.
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Drosophila melanogaster/fisiologia , Fenômenos Eletrofisiológicos , Rede Nervosa , Optogenética/métodos , Animais , Encéfalo/fisiologia , Relógios Circadianos , Drosophila melanogaster/citologia , Proteínas de Fluorescência Verde/genética , Neurônios/fisiologiaRESUMO
Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates that reduce fluorescence detection to near shot-noise levels. High-photon flux excitation can overcome photon-limited shot noise, but photobleaching and photodamage restrict the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, which is voltage imaging below the shot-noise limit. This framework involved developing positive-going voltage indicators with improved spike detection (SpikeyGi and SpikeyGi2); a two-photon microscope ('SMURF') for kilohertz frame rate imaging across a 0.4 mm × 0.4 mm field of view; and a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise-limited signals. Through these combined advances, we achieved simultaneous high-speed deep-tissue imaging of more than 100 densely labeled neurons over 1 hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.
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Microscopia , Neurônios , Camundongos , Animais , Neurônios/fisiologia , Algoritmos , CálcioRESUMO
To understand the operation of the olfactory system, it is essential to know how information is encoded in the olfactory bulb. We applied Shannon information theoretic methods to address this, with signals from up to 57 glomeruli simultaneously optically imaged from presynaptic inputs in glomeruli in the mouse dorsal (dOB) and lateral (lOB) olfactory bulb, in response to six exemplar pure chemical odors. We discovered that, first, the tuning of these signals from glomeruli to a set of odors is remarkably broad, with a mean sparseness of 0.83 and a mean signal correlation of 0.64. Second, both of these factors contribute to the low information that is available from the responses of even populations of many tens of glomeruli, which was only 1.35 bits across 33 glomeruli on average, compared with the 2.58 bits required to perfectly encode these six odors. Third, although there is considerable interest in the possibility of temporal encoding of stimulus including odor identity, the amount of information in the temporal aspects of the presynaptic glomerular responses was low (mean 0.11 bits) and, importantly, was redundant with respect to the information available from the rates. Fourth, the information from simultaneously recorded glomeruli asymptotes very gradually and nonlinearly, showing that glomeruli do not have independent responses. Fifth, the information from a population became available quite rapidly, within 100 ms of sniff onset, and the peak of the glomerular response was at 200 ms. Sixth, the information from the lOB was not additive with that of the dOB.NEW & NOTEWORTHY We report broad tuning and low odor information available across the lateral and dorsal bulb populations of glomeruli. Even though response latencies can be significantly predictive of stimulus identity, such contained very little information and none that was not redundant with information based on rate coding alone. Last, in line with the emerging notion of the important role of earliest stages of responses ("primacy"), we report a very rapid rise in information after each inhalation.
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Odorantes , Bulbo Olfatório , Camundongos , Animais , Bulbo Olfatório/fisiologia , Olfato/fisiologia , Condutos Olfatórios/fisiologiaRESUMO
The mammalian olfactory bulb (OB) plays an essential role in odor processing during the perception of smell. Optical imaging of the OB has proven to be a key tool in elucidating the spatial odor mapping and temporal dynamics that underlie higher-order odor processing. Much is known about the activation of olfactory sensory neuron (OSN) glomerular responses in the dorsal olfactory bulb (dOB) during odor presentation. However, the dorsal bulb provides access to only approximately 25% of all glomeruli, and little is known about how the lateral bulb functions during this critical process. Here, we report, for the first time, simultaneous measurements of OSN glomerular activity from both the dOB and the lateral olfactory bulb (lOB), thus describing odor-specific spatial mapping and the temporal dynamics of olfactory input to both the dorsal and lateral bulb. Odor responses in the lateral bulb tended to be most prominent in the dorso-lateral (D-L) region. Lateral glomeruli became active in a dorso-ventral (D-V) sequence upon odor inhalation, unlike the anterio-posterior (A-P) activity wave typical of the dorsal glomeruli. Across the entire D-L bulb, the spatial organization of these dynamics can be explained neither by the purely mechanosensitive dynamics (to breathing clean air) nor by the response amplitudes across glomeruli. Instead, these dynamics can be explained by a combination of zonal receptor distributions, associated OB projections, and air flow paths across the epithelium upon inhalation. Remarkably, we also found that a subset of OSN glomeruli in the lOB was highly sensitive to extranasal air pressure changes, a response type that has not been reported in dorsal glomeruli.
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Bulbo Olfatório/fisiologia , Percepção Olfatória/fisiologia , Animais , Mapeamento Encefálico , Feminino , Masculino , Mecanotransdução Celular , Camundongos Transgênicos , Odorantes , Bulbo Olfatório/diagnóstico por imagem , OlfatoRESUMO
Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50 mW mm-2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.
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Potenciais de Ação , Encéfalo/fisiologia , Drosophila melanogaster/metabolismo , Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador/métodos , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Animais , Encéfalo/citologia , Células Cultivadas , Fenômenos Eletrofisiológicos , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/fisiologia , Optogenética , Proteína Vermelha FluorescenteRESUMO
Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients toward preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations. In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis.
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Comportamento Animal/fisiologia , Drosophila melanogaster/fisiologia , Termorreceptores/fisiologia , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio , Gânglios/fisiologia , Larva/fisiologia , Locomoção/fisiologia , Optogenética , Sensação Térmica/fisiologiaRESUMO
Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (â¼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.
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Corantes Fluorescentes/análise , Hipocampo/química , Hipocampo/fisiologia , Proteínas Luminescentes/análise , Neurônios/química , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Microscopia de Fluorescência/métodos , Técnicas de Cultura de Órgãos/métodos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteína Vermelha FluorescenteRESUMO
Significance: Voltage imaging is a powerful tool for studying the dynamics of neuronal activities in the brain. However, voltage imaging data are fundamentally corrupted by severe Poisson noise in the low-photon regime, which hinders the accurate extraction of neuronal activities. Self-supervised deep learning denoising methods have shown great potential in addressing the challenges in low-photon voltage imaging without the need for ground truth, but usually suffer from the tradeoff between spatial and temporal performance. Aim: We present DeepVID v2, a novel self-supervised denoising framework with decoupled spatial and temporal enhancement capability to significantly augment low-photon voltage imaging. Approach: DeepVID v2 is built on our original DeepVID framework,1,2 which performs frame-based denoising by utilizing a sequence of frames around the central frame targeted for denoising to leverage temporal information and ensure consistency. The network further integrates multiple blind pixels in the central frame to enrich the learning of local spatial information. Additionally, DeepVID v2 introduces a new edge extraction branch to capture fine structural details in order to learn high spatial resolution information. Results: We demonstrate that DeepVID v2 is able to overcome the tradeoff between spatial and temporal performance, and achieve superior denoising capability in resolving both high-resolution spatial structures and rapid temporal neuronal activities. We further show that DeepVID v2 is able to generalize to different imaging conditions, including time-series measurements with various signal-to-noise ratios (SNRs) and in extreme low-photon conditions. Conclusions: Our results underscore DeepVID v2 as a promising tool for enhancing voltage imaging. This framework has the potential to generalize to other low-photon imaging modalities and greatly facilitate the study of neuronal activities in the brain.
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Optical neurotechnologies use light to interface with neurons and can monitor and manipulate neural activity with high spatial-temporal precision over large cortical extents. While there has been significant progress in miniaturizing microscope for head-mounted configurations, these existing devices are still very bulky and could never be fully implanted. Any viable translation of these technologies to human use will require a much more noninvasive, fully implantable form factor. Here, we leverage advances in microelectronics and heterogeneous optoelectronic packaging to develop a transformative, ultrathin, miniaturized device for bidirectional optical stimulation and recording: the subdural CMOS Optical Probe (SCOPe). By being thin enough to lie entirely within the subdural space of the primate brain, SCOPe defines a path for the eventual human translation of a new generation of brain-machine interfaces based on light.
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Genetically encoded voltage indicators (GEVIs) allow optical recordings of membrane potential changes in defined cell populations. Transgenic reporter animals that facilitate precise and repeatable targeting with high expression levels would further the use of GEVIs in the in vivo mammalian brain. However, the literature on developing and applying transgenic mouse lines as vehicles for GEVI expression is limited. Here we report the first in vivo experiments using a transgenic reporter mouse for the GEVI ArcLight, which utilizes a Cre/tTA dependent expression system (TIGRE 1.0). We developed two mouse lines with ArcLight expression restricted to either olfactory receptor neurons, or a subpopulation of interneurons located in the granule and glomerular layers in the olfactory bulb. The ArcLight expression in these lines was sufficient for in vivo imaging of odorant responses in single trials using epifluorescence and 2-photon imaging. The voltage responses were odor-specific and concentration-dependent, which supported earlier studies about perceptual transformations carried out by the bulb that used calcium sensors of neural activity. This study demonstrates that the ArcLight transgenic line is a flexible genetic tool that can be used to record the neuronal electrical activity of different cell types with a signal-to-noise ratio that is comparable to previous reports using viral transduction.
Assuntos
Técnicas Biossensoriais , Interneurônios/metabolismo , Proteínas Luminescentes/metabolismo , Potenciais da Membrana , Bulbo Olfatório/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagens com Corantes Sensíveis à Voltagem , Animais , Genes Reporter , Proteínas Luminescentes/genética , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Odorantes , Bulbo Olfatório/citologia , Percepção Olfatória , Proteínas Recombinantes de Fusão/genética , OlfatoRESUMO
Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR. Our indicators enable 0.4- to 1-kilohertz voltage recordings from >50 spiking neurons per field of view in awake mice and ~30-minute continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered brain state-dependent antagonism between neocortical somatostatin-expressing (SST+) and vasoactive intestinal peptide-expressing (VIP+) interneurons and contributions to hippocampal field potentials from cell ensembles with distinct axonal projections. By combining three mutually compatible indicators, we performed simultaneous triple-population imaging. These approaches will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution.
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Potenciais de Ação , Hipocampo , Imagem Molecular , Neurônios , Córtex Visual , Animais , Camundongos , Potenciais de Ação/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Interneurônios/fisiologia , Neurônios/classificação , Neurônios/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Imagem Molecular/métodos , Rodopsina/química , Rodopsina/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Córtex Visual/citologia , Córtex Visual/fisiologia , Fluorescência , Medições LuminescentesRESUMO
A central question about the brain is how information is processed by large populations of neurons embedded in intricate local networks. Answering this question requires not only monitoring functional dynamics of many neurons simultaneously, but also interpreting such activity patterns in the context of neuronal circuitry. Here, we introduce a versatile approach for loading Ca(2+) indicators in vivo by local electroporation. With this method, Ca(2+) imaging can be performed both at neuron population level and with exquisite subcellular resolution down to dendritic spines and axon boutons. This enabled mitral cell odor-evoked ensemble activity to be analyzed simultaneously with revealing their specific connectivity to different glomeruli. Colabeling of Purkinje cell dendrites and intersecting parallel fibers allowed Ca(2+) imaging of both presynaptic boutons and postsynaptic dendrites. This approach thus provides an unprecedented capability for in vivo visualizing active cell ensembles and tracing their underlying local neuronal circuits.
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Vias Aferentes/fisiologia , Encéfalo/citologia , Cálcio/metabolismo , Diagnóstico por Imagem/métodos , Rede Nervosa/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Dendritos/metabolismo , Dextranos/metabolismo , Estimulação Elétrica/métodos , Eletroporação , Potenciais Evocados/efeitos da radiação , Camundongos , Rede Nervosa/citologia , Neurônios/citologia , Odorantes , Vibrissas/inervaçãoRESUMO
We examined the effect of large, controlled whisker movements, delivered at a high speed, on the amplitude and spread of depolarization in the anesthetized mouse barrel cortex. The stimulus speed was varied between 1500 and 6000°/s and the extent of movement was varied between 4° and 16°. The rate of rise of the response was linearly related to the rate of rise of the stimulus. The initial spatial extent of cortical activation was also related to the rate of rise of the stimulus: that is, the faster the stimulus onset, the faster the rate of rise of the response, the larger the extent of cortex activated initially. The spatial extent of the response and the rate of rise of the response were not correlated with changes in the deflection amplitude. However, slower, longer lasting stimuli produced an Off response, making the actual extent of activation larger for the slowest rising stimuli. These results indicate that the spread of cortical activation depends on stimulus features.
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Neurônios/fisiologia , Estimulação Física , Córtex Somatossensorial/fisiologia , Vibrissas/fisiologia , Animais , Masculino , Camundongos , Estimulação Física/métodos , Imagens com Corantes Sensíveis à VoltagemRESUMO
Over the past decade, fluorescent proteins (FPs) have become ubiquitous tools in biological research. Yet, little is known about the natural function or evolution of this superfamily of proteins that originate from marine organisms. Using molecular phylogenetic analyses of 102 naturally occurring cyan fluorescent proteins, green fluorescent proteins, red fluorescent proteins, as well as the nonfluorescent (purple-blue) protein sequences (including new FPs from Lizard Island, Australia) derived from organisms with known geographic origin, we show that FPs consist of two distinct and novel regions that have evolved under opposite and sharply divergent evolutionary pressures. A central region is highly conserved, and although it contains the residues that form the chromophore, its evolution does not track with fluorescent color and evolves independently from the rest of the protein. By contrast, the regions enclosing this central region are under strong positive selection pressure to vary its sequence and yet segregate well with fluorescence color emission. We did not find a significant correlation between geographic location of the organism from which the FP was isolated and molecular evolution of the protein. These results define for the first time two distinct regions based on evolution for this highly compact protein. The findings have implications for more sophisticated bioengineering of this molecule as well as studies directed toward understanding the natural function of FPs.
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Evolução Molecular , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Sequência de Aminoácidos , Sequência Conservada , Glicoproteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Pyrosomes are tunicates in the phylum Chordata, which also contains vertebrates. Their gigantic blooms play important ecological and biogeochemical roles in oceans. Pyrosoma, meaning "fire-body", derives from their brilliant bioluminescence. The biochemistry of this light production is unknown, but has been hypothesized to be bacterial in origin. We found that mixing coelenterazine-a eukaryote-specific luciferin-with Pyrosoma atlanticum homogenate produced light. To identify the bioluminescent machinery, we sequenced P. atlanticum transcriptomes and found a sequence match to a cnidarian luciferase (RLuc). We expressed this novel luciferase (PyroLuc) and, combined with coelenterazine, it produced light. A similar gene was recently predicted from a bioluminescent brittle star, indicating that RLuc-like luciferases may have evolved convergently from homologous dehalogenases across phyla (Cnidaria, Echinodermata, and Chordata). This report indicates that a widespread gene may be able to functionally converge, resulting in bioluminescence across animal phyla, and describes and characterizes the first putative chordate luciferase.
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Luciferases/genética , Urocordados , Animais , Evolução Biológica , Cordados , Biologia Computacional , Evolução Molecular , Perfilação da Expressão Gênica , Imidazóis , Luminescência , Medições Luminescentes , Modelos Moleculares , Filogenia , Pirazinas , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Talin, an adaptor between integrin and the actin cytoskeleton at sites of cell adhesion, was recently found to be present at neuronal synapses, where its function remains unknown. Talin interacts with phosphatidylinositol-(4)-phosphate 5-kinase type Igamma, the major phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)]-synthesizing enzyme in brain. To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin-PIP kinase interaction and then examined their effects on synaptic structure. A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed. The endocytic defect included an accumulation of clathrin-coated pits with wide necks, as previously observed after perturbing actin at these synapses. Thus, the interaction of PIP kinase with talin in presynaptic compartments provides a mechanism to coordinate PI(4,5)P(2) synthesis, actin dynamics, and endocytosis, and further supports a functional link between actin and clathrin-mediated endocytosis.
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Actinas/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia , Talina/fisiologia , Animais , Western Blotting , Adesão Celular , Clatrina/metabolismo , Endocitose , Glutationa Transferase/metabolismo , Humanos , Lampreias , Metabolismo dos Lipídeos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Peptídeos/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Talina/química , Talina/metabolismo , Fatores de TempoRESUMO
It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.
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Actinas/metabolismo , Lampreias/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Endocitose , Microinjeções , Microscopia Imunoeletrônica , Modelos Biológicos , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura , Sinapses/química , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/ultraestruturaRESUMO
Genetically encoded optical indicators of neuronal activity enable unambiguous recordings of input-output activity patterns from identified cells in intact circuits. Among them, genetically encoded voltage indicators (GEVIs) offer additional advantages over calcium indicators as they are direct sensors of membrane potential and can adeptly report subthreshold events and hyperpolarization. Here, we outline the major GEVI designs and give an account of properties that need to be carefully optimized during indicator engineering. While designing the ideal GEVI, one should keep in mind aspects such as membrane localization, signal size, signal-to-noise ratio, kinetics and voltage dependence of optical responses. Using ArcLight and derivatives as prototypes, we delineate how a probe should be optimized for the former properties and developed along other areas in a need-based manner. Finally, we present an overview of the GEVI engineering process and lend an insight into their discovery, delivery and diagnosis.