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1.
Curr Biol ; 33(14): 2912-2924.e5, 2023 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-37379842

RESUMO

Internal predictions about the sensory consequences of self-motion, encoded by corollary discharge, are ubiquitous in the animal kingdom, including for fruit flies, dragonflies, and humans. In contrast, predicting the future location of an independently moving external target requires an internal model. With the use of internal models for predictive gaze control, vertebrate predatory species compensate for their sluggish visual systems and long sensorimotor latencies. This ability is crucial for the timely and accurate decisions that underpin a successful attack. Here, we directly demonstrate that the robber fly Laphria saffrana, a specialized beetle predator, also uses predictive gaze control when head tracking potential prey. Laphria uses this predictive ability to perform the difficult categorization and perceptual decision task of differentiating a beetle from other flying insects with a low spatial resolution retina. Specifically, we show that (1) this predictive behavior is part of a saccade-and-fixate strategy, (2) the relative target angular position and velocity, acquired during fixation, inform the subsequent predictive saccade, and (3) the predictive saccade provides Laphria with additional fixation time to sample the frequency of the prey's specular wing reflections. We also demonstrate that Laphria uses such wing reflections as a proxy for the wingbeat frequency of the potential prey and that consecutively flashing LEDs to produce apparent motion elicits attacks when the LED flicker frequency matches that of the beetle's wingbeat cycle.


Assuntos
Besouros , Crocus , Odonatos , Humanos , Animais , Movimentos Sacádicos , Tomada de Decisões
2.
Nat Methods ; 20(6): 824-835, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37069271

RESUMO

BigNeuron is an open community bench-testing platform with the goal of setting open standards for accurate and fast automatic neuron tracing. We gathered a diverse set of image volumes across several species that is representative of the data obtained in many neuroscience laboratories interested in neuron tracing. Here, we report generated gold standard manual annotations for a subset of the available imaging datasets and quantified tracing quality for 35 automatic tracing algorithms. The goal of generating such a hand-curated diverse dataset is to advance the development of tracing algorithms and enable generalizable benchmarking. Together with image quality features, we pooled the data in an interactive web application that enables users and developers to perform principal component analysis, t-distributed stochastic neighbor embedding, correlation and clustering, visualization of imaging and tracing data, and benchmarking of automatic tracing algorithms in user-defined data subsets. The image quality metrics explain most of the variance in the data, followed by neuromorphological features related to neuron size. We observed that diverse algorithms can provide complementary information to obtain accurate results and developed a method to iteratively combine methods and generate consensus reconstructions. The consensus trees obtained provide estimates of the neuron structure ground truth that typically outperform single algorithms in noisy datasets. However, specific algorithms may outperform the consensus tree strategy in specific imaging conditions. Finally, to aid users in predicting the most accurate automatic tracing results without manual annotations for comparison, we used support vector machine regression to predict reconstruction quality given an image volume and a set of automatic tracings.


Assuntos
Benchmarking , Microscopia , Microscopia/métodos , Imageamento Tridimensional/métodos , Neurônios/fisiologia , Algoritmos
3.
J Exp Biol ; 226(4)2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36695720

RESUMO

The ability to visualize small moving objects is vital for the survival of many animals, as these could represent predators or prey. For example, predatory insects, including dragonflies, robber flies and killer flies, perform elegant, high-speed pursuits of both biological and artificial targets. Many non-predatory insects, including male hoverflies and blowflies, also pursue targets during territorial or courtship interactions. To date, most hoverfly pursuits have been studied outdoors. To investigate hoverfly (Eristalis tenax) pursuits under more controlled settings, we constructed an indoor arena that was large enough to encourage naturalistic behavior. We presented artificial beads of different sizes, moving at different speeds, and filmed pursuits with two cameras, allowing subsequent 3D reconstruction of the hoverfly and bead position as a function of time. We show that male E. tenax hoverflies are unlikely to use strict heuristic rules based on angular size or speed to determine when to start pursuit, at least in our indoor setting. We found that hoverflies pursued faster beads when the trajectory involved flying downwards towards the bead. Furthermore, we show that target pursuit behavior can be broken down into two stages. In the first stage, the hoverfly attempts to rapidly decreases the distance to the target by intercepting it at high speed. During the second stage, the hoverfly's forward speed is correlated with the speed of the bead, so that the hoverfly remains close, but without catching it. This may be similar to dragonfly shadowing behavior, previously coined 'motion camouflage'.


Assuntos
Dípteros , Odonatos , Animais , Masculino , Insetos , Territorialidade , Comportamento Predatório
4.
Curr Opin Insect Sci ; 52: 100914, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35346895

RESUMO

Under strong selective pressure for survival, image-forming vision set off an ongoing predatory arms race 500 million years ago. Since then, and particularly so in the arthropods, predatory behavior has driven a myriad of eye adaptations that increase visual performance. In this review, we provide examples of how different arthropod predators have achieved improvements in key visual features such as spatial and temporal resolution of their retina. We then describe morphological, neural and behavioral strategies used by animals in this group to gather crucial information about the prey, such as its distance, velocity and size. We also highlight the importance of head and body tracking movements to aid in categorizing the potential prey, and briefly mention the ongoing work on the sensorimotor transformations necessary for target interception.


Assuntos
Artrópodes , Comportamento Predatório , Animais , Retina , Visão Ocular
5.
J Exp Biol ; 225(4)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35168251

RESUMO

The miniature robber fly Holcocephala fusca intercepts its targets with behaviour that is approximated by the proportional navigation guidance law. During predatory trials, we challenged the interception of H. fusca performance by placing a large object in its potential flight path. In response, H. fusca deviated from the path predicted by pure proportional navigation, but in many cases still eventually contacted the target. We show that such flight deviations can be explained as the output of two competing navigational systems: pure-proportional navigation and a simple obstacle avoidance algorithm. Obstacle avoidance by H. fusca is here described by a simple feedback loop that uses the visual expansion of the approaching obstacle to mediate the magnitude of the turning-away response. We name the integration of this steering law with proportional navigation 'combined guidance'. The results demonstrate that predatory intent does not operate a monopoly on the fly's steering when attacking a target, and that simple guidance combinations can explain obstacle avoidance during interceptive tasks.


Assuntos
Voo Animal , Comportamento Predatório , Animais , Voo Animal/fisiologia
6.
Curr Biol ; 30(4): 645-656.e4, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-31956029

RESUMO

Akin to all damselflies, Calopteryx (family Calopterygidae), commonly known as jewel wings or demoiselles, possess dichoptic (separated) eyes with overlapping visual fields of view. In contrast, many dragonfly species possess holoptic (dorsally fused) eyes with limited binocular overlap. We have here compared the neuronal correlates of target tracking between damselfly and dragonfly sister lineages and linked these changes in visual overlap to pre-motor neural adaptations. Although dragonflies attack prey dorsally, we show that demoiselles attack prey frontally. We identify demoiselle target-selective descending neurons (TSDNs) with matching frontal visual receptive fields, anatomically and functionally homologous to the dorsally positioned dragonfly TSDNs. By manipulating visual input using eyepatches and prisms, we show that moving target information at the pre-motor level depends on binocular summation in demoiselles. Consequently, demoiselles encode directional information in a binocularly fused frame of reference such that information of a target moving toward the midline in the left eye is fused with information of the target moving away from the midline in the right eye. This contrasts with dragonfly TSDNs, where receptive fields possess a sharp midline boundary, confining responses to a single visual hemifield in a sagittal frame of reference (i.e., relative to the midline). Our results indicate that, although TSDNs are conserved across Odonata, their neural inputs, and thus the upstream organization of the target tracking system, differ significantly and match divergence in eye design and predatory strategies. VIDEO ABSTRACT.


Assuntos
Voo Animal , Odonatos/fisiologia , Comportamento Predatório/fisiologia , Campos Visuais/fisiologia , Animais
7.
J Exp Biol ; 223(Pt 4)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-31988162

RESUMO

Mantis shrimp strikes are one of the fastest animal movements, despite their occurrence in a water medium with viscous drag. Since the strike is produced by a latch-mediated spring-actuated system and not directly driven by muscle action, we predicted that strikes performed in air would be faster than underwater as a result of reduction in the medium's drag. Using high-speed video analysis of stereotyped strikes elicited from Squilla mantis, we found the exact opposite: strikes are much slower and less powerful in air than in water. S. mantis strikes in air have a similar mass and performance to latch-mediated spring-actuated jumps in locusts, suggesting a potential threshold for the energetics of a 1-2 g limb rotating in air. Drag forces induced by the media may be a key feature in the evolution of mantis shrimp strikes and provide a potential target for probing the braking system of these extremely fast movements.


Assuntos
Comportamento Animal/fisiologia , Crustáceos/fisiologia , Movimento , Ar , Animais , Fenômenos Biomecânicos , Feminino , Masculino , Comportamento Predatório/fisiologia , Gravação em Vídeo , Água
9.
J R Soc Interface ; 15(147)2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333249

RESUMO

When aiming to capture a fast-moving target, animals can follow it until they catch up, or try to intercept it. In principle, interception is the more complicated strategy, but also more energy efficient. To study whether simple feedback controllers can explain interception behaviours by animals with miniature brains, we have reconstructed and studied the predatory flights of the robber fly Holcocephala fusca and killer fly Coenosia attenuata Although both species catch other aerial arthropods out of the air, Holcocephala contrasts prey against the open sky, while Coenosia hunts against clutter and at much closer range. Thus, their solutions to this target catching task may differ significantly. We reconstructed in three dimensions the flight trajectories of these two species and those of the presented targets they were attempting to intercept. We then tested their recorded performances against simulations. We found that both species intercept targets on near time-optimal courses. To investigate the guidance laws that could underlie this behaviour, we tested three alternative control systems (pure pursuit, deviated pursuit and proportional navigation). Only proportional navigation explains the timing and magnitude of fly steering responses, but with differing gain constants and delays for each fly species. Holcocephala uses a dimensionless navigational constant of N ≈ 3 with a time delay of ≈28 ms to intercept targets over a comparatively long range. This constant is optimal, as it minimizes the control effort required to hit the target. In contrast, Coenosia uses a constant of N ≈ 1.5 with a time delay of ≈18 ms, this setting may allow Coenosia to cope with the extremely high line-of-sight rotation rates, which are due to close target proximity, and thus prevent overcompensation of steering. This is the first clear evidence of interception supported by proportional navigation in insects. This work also demonstrates how by setting different gains and delays, the same simple feedback controller can yield the necessary performance in two different environments.


Assuntos
Dípteros/fisiologia , Voo Animal/fisiologia , Comportamento Predatório/fisiologia , Animais , Modelos Biológicos
10.
J Neurosci ; 38(50): 10725-10733, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30373766

RESUMO

For many animals, target motion carries high ecological significance as this may be generated by a predator, prey, or potential mate. Indeed, animals whose survival depends on early target detection are often equipped with a sharply tuned visual system, yielding robust performance in challenging conditions. For example, many fast-flying insects use visual cues for identifying targets, such as prey (e.g., predatory dragonflies and robberflies) or conspecifics (e.g., nonpredatory hoverflies), and can often do so against self-generated background optic flow. Supporting these behaviors, the optic lobes of insects that pursue targets harbor neurons that respond robustly to the motion of small moving objects, even when displayed against syn-directional background clutter. However, in diptera, the encoding of target information by the descending neurons, which are more directly involved in generating the behavioral output, has received less attention. We characterized target-selective neurons by recording in the ventral nerve cord of male and female predatory Holcocephala fusca robberflies and of male nonpredatory Eristalis tenax hoverflies. We show that both species have dipteran target-selective descending neurons that only respond to target motion if the background is stationary or moving slowly, moves in the opposite direction, or has un-naturalistic spatial characteristics. The response to the target is suppressed when background and target move at similar velocities, which is strikingly different to the response of target neurons in the optic lobes. As the neurons we recorded from are premotor, our findings affect our interpretation of the neurophysiology underlying target-tracking behaviors.SIGNIFICANCE STATEMENT Many animals use sensory cues to detect moving targets that may represent predators, prey, or conspecifics. For example, birds of prey show superb sensitivity to the motion of small prey, and intercept these at high speeds. In a similar manner, predatory insects visually track moving prey, often against cluttered backgrounds. Accompanying this behavior, the brains of insects that pursue targets contain neurons that respond exclusively to target motion. We here show that dipteran insects also have target-selective descending neurons in the part of their nervous system that corresponds to the vertebrate spinal cord. Surprisingly, and in contrast to the neurons in the brain, these premotor neurons are inhibited by background patterns moving in the same direction as the target.


Assuntos
Percepção de Movimento/fisiologia , Neurônios/fisiologia , Lobo Óptico de Animais não Mamíferos/fisiologia , Estimulação Luminosa/métodos , Comportamento Predatório/fisiologia , Campos Visuais/fisiologia , Animais , Dípteros , Feminino , Masculino , Odonatos , Especificidade da Espécie
11.
iScience ; 1: 24-34, 2018 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-30058000

RESUMO

The color and pattern changing abilities of octopus, squid, and cuttlefish via chromatophore neuromuscular organs are unparalleled. Cuttlefish and octopuses also have a unique muscular hydrostat system in their skin. When this system is expressed, dermal bumps called papillae disrupt body shape and imitate the fine texture of surrounding objects, yet the control system is unknown. Here we report for papillae: (1) the motoneurons and the neurotransmitters that control activation and relaxation, (2) a physiologically fast expression and retraction system, and (3) a complex of smooth and striated muscles that enables long-term expression of papillae through sustained tension in the absence of neural input. The neural circuits controlling acute shape-shifting skin papillae in cuttlefish show homology to the iridescence circuits in squids. The sustained tension in papillary muscles for long-term camouflage utilizes muscle heterogeneity and points toward the existence of a "catch-like" mechanism that would reduce the necessary energy expenditure.

12.
J Exp Biol ; 221(Pt 10)2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29720383

RESUMO

On warm sunny days, female hoverflies are often observed feeding from a wide range of wild and cultivated flowers. In doing so, hoverflies serve a vital role as alternative pollinators, and are suggested to be the most important pollinators after bees and bumblebees. Unless the flower hoverflies are feeding from is large, they do not readily share the space with other insects, but instead opt to leave if another insect approaches. We used high-speed videography followed by 3D reconstruction of flight trajectories to quantify how female Eristalis hoverflies respond to approaching bees, wasps and two different hoverfly species. We found that, in 94% of the interactions, the occupant female left the flower when approached by another insect. We found that compared with spontaneous take-offs, the occupant hoverfly's escape response was performed at ∼3 times higher speed (spontaneous take-off at 0.2±0.05 m s-1 compared with 0.55±0.08 m s-1 when approached by another Eristalis). The hoverflies tended to take off upward and forward, while taking the incomer's approach angle into account. Intriguingly, we found that, when approached by wasps, the occupant Eristalis took off at a higher speed and when the wasp was further away. This suggests that feeding hoverflies may be able to distinguish these predators, demanding impressive visual capabilities. Our results, including quantification of the visual information available before occupant take-off, provide important insight into how freely behaving hoverflies perform escape responses from competitors and predators (e.g. wasps) in the wild.


Assuntos
Dípteros/fisiologia , Comportamento Alimentar , Visão Ocular , Animais , Abelhas , Feminino , Voo Animal , Flores , Comportamento Predatório , Gravação em Vídeo , Vespas
13.
Curr Biol ; 27(6): 854-859, 2017 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-28286000

RESUMO

Our visual system allows us to rapidly identify and intercept a moving object. When this object is far away, we base the trajectory on the target's location relative to an external frame of reference [1]. This process forms the basis for the constant bearing angle (CBA) model, a reactive strategy that ensures interception since the bearing angle, formed between the line joining pursuer and target (called the range vector) and an external reference line, is held constant [2-4]. The CBA model may be a fundamental and widespread strategy, as it is also known to explain the interception trajectories of bats and fish [5, 6]. Here, we show that the aerial attack of the tiny robber fly Holcocephala fusca is consistent with the CBA model. In addition, Holcocephala fusca displays a novel proactive strategy, termed "lock-on" phase, embedded with the later part of the flight. We found the object detection threshold for this species to be 0.13°, enabled by an extremely specialized, forward pointing fovea (∼5 ommatidia wide, interommatidial angle Δφ = 0.28°, photoreceptor acceptance angle Δρ = 0.27°). This study furthers our understanding of the accurate performance that a miniature brain can achieve in highly demanding sensorimotor tasks and suggests the presence of equivalent mechanisms for target interception across a wide range of taxa. VIDEO ABSTRACT.


Assuntos
Dípteros/fisiologia , Percepção de Movimento , Acuidade Visual , Animais , Desempenho Psicomotor
14.
Curr Opin Neurobiol ; 41: 122-128, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27662056

RESUMO

Motion vision provides important cues for many tasks. Flying insects, for example, may pursue small, fast moving targets for mating or feeding purposes, even when these are detected against self-generated optic flow. Since insects are small, with size-constrained eyes and brains, they have evolved to optimize their optical, neural and behavioral target visualization solutions. Indeed, even if evolutionarily distant insects display different pursuit strategies, target neuron physiology is strikingly similar. Furthermore, the coarse spatial resolution of the insect compound eye might actually be beneficial when it comes to detection of moving targets. In conclusion, tiny insects show higher than expected performance in target visualization tasks.


Assuntos
Comportamento Animal/fisiologia , Insetos/fisiologia , Animais , Percepção de Movimento/fisiologia , Neurônios/fisiologia , Visão Ocular/fisiologia
15.
Brain Behav Evol ; 86(1): 28-37, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26398293

RESUMO

Predatory animals have evolved to optimally detect their prey using exquisite sensory systems such as vision, olfaction and hearing. It may not be so surprising that vertebrates, with large central nervous systems, excel at predatory behaviors. More striking is the fact that many tiny insects, with their miniscule brains and scaled down nerve cords, are also ferocious, highly successful predators. For predation, it is important to determine whether a prey is suitable before initiating pursuit. This is paramount since pursuing a prey that is too large to capture, subdue or dispatch will generate a substantial metabolic cost (in the form of muscle output) without any chance of metabolic gain (in the form of food). In addition, during all pursuits, the predator breaks its potential camouflage and thus runs the risk of becoming prey itself. Many insects use their eyes to initially detect and subsequently pursue prey. Dragonflies, which are extremely efficient predators, therefore have huge eyes with relatively high spatial resolution that allow efficient prey size estimation before initiating pursuit. However, much smaller insects, such as killer flies, also visualize and successfully pursue prey. This is an impressive behavior since the small size of the killer fly naturally limits the neural capacity and also the spatial resolution provided by the compound eye. Despite this, we here show that killer flies efficiently pursue natural (Drosophila melanogaster) and artificial (beads) prey. The natural pursuits are initiated at a distance of 7.9 ± 2.9 cm, which we show is too far away to allow for distance estimation using binocular disparities. Moreover, we show that rather than estimating absolute prey size prior to launching the attack, as dragonflies do, killer flies attack with high probability when the ratio of the prey's subtended retinal velocity and retinal size is 0.37. We also show that killer flies will respond to a stimulus of an angular size that is smaller than that of the photoreceptor acceptance angle, and that the predatory response is strongly modulated by the metabolic state. Our data thus provide an exciting example of a loosely designed matched filter to Drosophila, but one which will still generate successful pursuits of other suitable prey.


Assuntos
Tomada de Decisões/fisiologia , Insetos/fisiologia , Comportamento Predatório/fisiologia , Percepção de Tamanho/fisiologia , Comportamento Espacial , Animais , Estimulação Luminosa , Probabilidade , Fatores de Tempo , Percepção do Tempo , Gravação em Vídeo
16.
Nat Commun ; 5: 4342, 2014 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-25014658

RESUMO

Three-dimensional (3D) bioimaging, visualization and data analysis are in strong need of powerful 3D exploration techniques. We develop virtual finger (VF) to generate 3D curves, points and regions-of-interest in the 3D space of a volumetric image with a single finger operation, such as a computer mouse stroke, or click or zoom from the 2D-projection plane of an image as visualized with a computer. VF provides efficient methods for acquisition, visualization and analysis of 3D images for roundworm, fruitfly, dragonfly, mouse, rat and human. Specifically, VF enables instant 3D optical zoom-in imaging, 3D free-form optical microsurgery, and 3D visualization and annotation of terabytes of whole-brain image volumes. VF also leads to orders of magnitude better efficiency of automated 3D reconstruction of neurons and similar biostructures over our previous systems. We use VF to generate from images of 1,107 Drosophila GAL4 lines a projectome of a Drosophila brain.


Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microcirurgia/métodos , Interface Usuário-Computador , Animais , Encéfalo/citologia , Caenorhabditis elegans , Drosophila , Pulmão/citologia , Camundongos , Modelos Animais , Células Musculares/citologia , Neurônios/citologia
17.
Curr Biol ; 23(15): R655-6, 2013 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-23928083

RESUMO

Visual systems adapt rapidly to objects moving repeatedly within the visual field, because such objects are likely irrelevant. In the crab, the neural switch for such adaptation has been found to take place at a surprisingly early stage of the visual processing pathway.


Assuntos
Comportamento Animal/fisiologia , Braquiúros/fisiologia , Plasticidade Neuronal , Neurônios/fisiologia , Lobo Óptico de Animais não Mamíferos/citologia , Animais
18.
BMC Bioinformatics ; 14: 93, 2013 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-23497429

RESUMO

BACKGROUND: Automatic 3D digital reconstruction (tracing) of neurons embedded in noisy microscopic images is challenging, especially when the cell morphology is complex. RESULTS: We have developed a novel approach, named DF-Tracing, to tackle this challenge. This method first extracts the neurite signal (foreground) from a noisy image by using anisotropic filtering and automated thresholding. Then, DF-Tracing executes a coupled distance-field (DF) algorithm on the extracted foreground neurite signal and reconstructs the neuron morphology automatically. Two distance-transform based "force" fields are used: one for "pressure", which is the distance transform field of foreground pixels (voxels) to the background, and another for "thrust", which is the distance transform field of the foreground pixels to an automatically determined seed point. The coupling of these two force fields can "push" a "rolling ball" quickly along the skeleton of a neuron, reconstructing the 3D cell morphology. CONCLUSION: We have used DF-Tracing to reconstruct the intricate neuron structures found in noisy image stacks, obtained with 3D laser microscopy, of dragonfly thoracic ganglia. Compared to several previous methods, DF-Tracing produces better reconstructions.


Assuntos
Imageamento Tridimensional/métodos , Neurônios/citologia , Algoritmos , Animais , Anisotropia , Microscopia Confocal , Neuritos/ultraestrutura , Odonatos/citologia
19.
Proc Natl Acad Sci U S A ; 110(2): 696-701, 2013 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-23213224

RESUMO

Intercepting a moving object requires prediction of its future location. This complex task has been solved by dragonflies, who intercept their prey in midair with a 95% success rate. In this study, we show that a group of 16 neurons, called target-selective descending neurons (TSDNs), code a population vector that reflects the direction of the target with high accuracy and reliability across 360°. The TSDN spatial (receptive field) and temporal (latency) properties matched the area of the retina where the prey is focused and the reaction time, respectively, during predatory flights. The directional tuning curves and morphological traits (3D tracings) for each TSDN type were consistent among animals, but spike rates were not. Our results emphasize that a successful neural circuit for target tracking and interception can be achieved with few neurons and that in dragonflies this information is relayed from the brain to the wing motor centers in population vector form.


Assuntos
Voo Animal/fisiologia , Percepção de Movimento/fisiologia , Odonatos/fisiologia , Comportamento Predatório/fisiologia , Neurônios Retinianos/fisiologia , Animais , Isoquinolinas , Microscopia Confocal , Modelos Neurológicos , Condução Nervosa/fisiologia , Estimulação Luminosa , Tempo de Reação , Neurônios Retinianos/citologia , Temperatura , Campos Visuais/fisiologia
20.
Cold Spring Harb Protoc ; 2012(9): 969-83, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22949711

RESUMO

Neuroscience researchers have long sought methods to describe the neural connectivity of the circuits responsible for specific behaviors. One major obstacle is scale: Neural spines can be <1 µm in diameter, but axons can range from millimeters to centimeters (or larger) in length, making tissue imaging and neuron reconstruction a challenging task. New tissue-clearing agents and long-working-distance objectives offer improved imaging conditions, and here we present a complete protocol for invertebrate tissue that uses these advances. In this protocol, tissue-processing steps previously published in separate articles are combined with recent advances in confocal imaging to visualize invertebrate tissue samples that are >500 µm thick and contain dye-filled neurons. The steps describe dye filling, fixing, antibody labeling, clearing, whole tissue mounting, and confocal imaging with matched refractive indexes. Thus, manual sectioning or "flipping" the tissue to image the whole volume is not required. With matched refractive indexes, loss of resolution and signal is avoided. Tissue volumes are imaged in one stack and nonlinear deformations caused by tissue flipping are prevented. We apply the protocol to whole dragonfly thoracic ganglia (2 × 1 × 0.6 mm) and cephalopod skin samples (20 × 2 × 0.6 mm) with minimal tissue deformation. The resulting images will be used to develop a three-dimensional connectivity atlas of dragonfly ganglia and cephalopod skin innervation. This protocol can be applied to other invertebrate species, and has the advantage that it avoids problems with antigen specificity.


Assuntos
Invertebrados/citologia , Microscopia Confocal/métodos , Neurônios/citologia , Coloração e Rotulagem/métodos , Animais , Processamento de Imagem Assistida por Computador/métodos
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