Name: techniques for investigating the anatomy of the ant visual system
Uploaded: 2017-11-27T15:00:00.000+00:00
Duration: 8 min 56 s
Description: Watch this Scientific Journal Video about Techniques for Investigating the Anatomy of the Ant Visual System at JoVE.com

Estamos gratos a Jochen Zeil, Paul Cooper e Birgit Greiner por compartilhar seus conhecimentos em anatomia de insetos e para várias das técnicas demonstrando descrevemos aqui. Estamos gratos aos funcionários talentosos e apoio do centro de microscopia avançada no ANU e a unidade de microscopia em MQU. Este trabalho foi apoiado por uma bolsa de pós-graduação para FRE e subsídios do Conselho australiano de pesquisa (DE120100019, FT140100221, DP150101172).

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Os autores declaram não interesses concorrentes.

O conjunto de métodos indicados acima permitem uma investigação eficaz sobre o sistema óptico de formigas e outros insetos. Estas técnicas informam nossa compreensão da resolução de amostragem, sensibilidade óptica e potencial sensibilidade de polarização do olho a ser estudado. Esse conhecimento fornece uma base importante para a investigação fisiológica e comportamental em suas capacidades visuais. Além disso, enquanto os métodos detalhados aqui concentraram-se em sistemas visuais de formiga, estas téc...

Visão é uma modalidade sensorial importante para a maioria dos animais. Visão é especialmente crucial no contexto de navegação para identificar objetivos, estabelecendo e aderindo às rotas e obter informações bússola1,2. Insetos detectam informação visual usando um par de olhos compostos e, em alguns casos, uma a três colocados dorsalmente simples olhos chamados ocelos3,4,5.
Os olhos das formigas são de particular interesse porque, enquanto as formigas são maravilhosamente diversificadas, eles conservar algumas características chaves em toda a espécie. Apesar da dramática variação no tamanho, anatomia e ecologia, a grande maioria das espécies é eussociais e vive em colônias; Como resultado, diferentes espécies enfrentam desafios visuais similares em termos de navegação e para trás entre um lugar central e recursos. Através de formigas a mesma bauplan olho básico pode ser observado nos animais que variam de 0,5-26 mm de comprimento, de exclusivamente diurnos a espécie estritamente noturno e de lento andar subterrâneo para saltar predadores visual6,7, 8,9,10. Todas estas diferenças de escalonamento em ecologia e comportamento dão origem a inúmeras permutações das estruturas básicas de olho mesmo para se adequar a diferentes ambientes, estilos de vida e corpo-tamanhos11,12. Como consequência, estudar a ecologia visual de formigas fornece um verdadeiro tesouro de possibilidades para o investigador determinado.
Compreender o sistema visual de insetos é essencial na obtenção de uma visão sobre suas capacidades comportamentais. Isto é evidente de estudos integrativo que combinam muito bem a anatomia com a ecologia e comportamento de um grande sucesso em alguns grupos de insetos (por exemplo, referências13,14,15,16, 17). embora o campo da formiga navegação e comportamento de formigas em geral tem sido muito bem sucedido, muito pouca ênfase foi colocada na visão de formiga fora algumas espécies selecionadas. Aqui, nós irá elaborar sobre as técnicas envolvidas no projeto de olho de formigas a investigar. Enquanto vamos nos concentrar em formigas, estas técnicas podem ser aplicadas, com pequenas modificações, para outros insetos, também.

<table><tbody><tr><td>Ant</td><td>Myrmecia midas</td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica M205 FA</td><td></td><td></td></tr><tr><td>Sputter coater</td><td>Pro Sci Tech</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Petri dish</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica MZ6</td><td></td><td></td></tr><tr><td>Insect Pin</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Colourless nail polish</td><td>Non branded: from any cosmetic store</td><td></td><td></td></tr><tr><td>Glass slide</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Razor blade</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Foreceps</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Cover slip</td><td>ProSciTech</td><td></td><td></td></tr><tr><td>Compound microscope</td><td>Leica DM5000 B</td><td></td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Paraformalydehyde</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>di-Sodium Hydrogen phosphate (Na2HPO4)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Potassium di-Hydrogen Phosphate (KH2PO4)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Osmium tetroxide</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Araldite Epoxy Resin</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Pasteur pipette</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Toluidie Blue</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Hotplate</td><td>Riechert HK120</td><td></td><td></td></tr></tbody></table>

techniques for investigating the anatomy of the ant visual system

Este artigo descreve um conjunto de técnicas em microscopia de luz (LM) e microscopia de elétron (EM) que pode ser usada para estudar a anatomia interna e externa do olho de insetos. Estes incluem técnicas histológicas tradicionais otimizado para trabalhar em olhos de formiga e adaptado para trabalhar em conjunto com outras técnicas, como a microscopia eletrônica de transmissão (TEM) e microscopia eletrônica de varredura (MEV). Essas técnicas, embora muito útil, podem ser difícil para a microscopista iniciante, então grande ênfase foi colocada neste artigo sobre solução de problemas e otimização para espécimes diferentes. Podemos fornecer informações sobre técnicas de imagem para o espécime inteiro (foto-microscopia e SEM) e discutir as suas vantagens e desvantagens. Destaca-se a técnica utilizada na determinação de diâmetros de lente para o olho inteiro e discutir novas técnicas para melhoria. Por fim, discutimos as técnicas envolvidas na preparação de amostras para LM e TEM, corte, coloração e estas amostras de imagem. Vamos discutir os obstáculos que um pode vir do outro lado quando preparar amostras e melhor forma de navegar ao redor deles.

Os métodos descritos aqui permitem o estudo detalhado da simples e olhos compostos das formigas. A vista dorsal da cabeça usando técnicas de fotomicrografia de Z-pilha de imagem permite obter uma visão geral do layout do sistema visual (Figura 1). Esta é uma boa preparação para as dissecções e para determinar o ângulo de corte desejado. Esta técnica também é útil para a tomada de medidas tais como a largura da cabeça, comprimento do olho e diâ...

Watch this Scientific Journal Video about Techniques for Investigating the Anatomy of the Ant Visual System at JoVE.com

Techniques for Investigating the Anatomy of the Ant Visual System

Este artigo descreve um conjunto de técnicas de luz e microscopia eletrônica de varredura para estudar a anatomia interna e externa do olho de insetos. Estes incluem várias técnicas tradicionais otimizadas para trabalhar em olhos de formiga, detalhados de solução de problemas e sugestões para otimização de espécimes diferentes e regiões de interesse.

Técnicas para investigar a anatomia do sistema Visual de formiga

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

techniques-for-investigating-the-anatomy-of-the-ant-visual-system

Research

JoVE Journal

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13.2K visualizações.  Australian National University. Este artigo descreve um conjunto de técnicas de luz e microscopia eletrônica de varredura para estudar a anatomia interna e externa do olho de insetos. Estes incluem várias técnicas tradicionais otimizadas para trabalhar em olhos de formiga, detalhados de solução de problemas e sugestões para otimização de espécimes diferentes e regiões de interesse.

Vídeo: Techniques for Investigating the Anatomy of the Ant Visual System

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Visual condicionamento clássico em madeira formigas

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

Comportamento

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

A entrada para o cérebro: Dissecando o olho Primaz

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Biologia

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Condicionado tátil e Análise do Movimento de estratégias de amostragem antenais de abelhas (<em&gt; Apis mellifera</em&gt; L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Neurociência

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparação de Desenvolvimento e Adultos Drosophila Brains e retina de imagens ao vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

Dissecção, imuno-histoquímica e montagem de cérebros de larvas e adultos de Drosophila para visualização do lobo óptico

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

Biologia do Desenvolvimento

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

Um sistema de explant para estudos de imagem de lapso de tempo da montagem do circuito olfativo em Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Analisando dendríticas Morfologia em Colunas e Camadas

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

<meta charset="utf8"></head><body>Remodelação sináptica induzida pela experiência olfativa no cérebro juvenil de Drosophila

Experience-Dependent Remodeling of Juvenile Brain Olfactory Sensory Neuron Synaptic Connectivity in an Early-Life Critical Period

Early-life olfactory sensory experience induces dramatic synaptic glomeruli remodeling in the Drosophila juvenile brain, which is experientially dose-dependent, temporally restricted, and transiently reversible only in a short, well-defined critical period. The directionality of brain circuit synaptic connectivity remodeling is determined by the specific odorant acting on the respondent receptor class of olfactory sensory neurons. In general, each neuron class expresses only a single odorant receptor and innervates a single olfactory synaptic glomerulus. In the Drosophila genetic model, the full array of olfactory glomeruli has been precisely mapped by odorant responsiveness and behavioral output. Ethyl butyrate (EB) odorant activates Or42a receptor neurons innervating the VM7 glomerulus. During the early-life critical period, EB experience drives dose-dependent synapse elimination in the Or42a olfactory sensory neurons. Timed periods of dosed EB odorant exposure allow investigation of experience-dependent circuit connectivity pruning in juvenile brain. Confocal microscopy imaging of antennal lobe synaptic glomeruli is done with Or42a receptor-driven transgenic markers that provide quantification of synapse number and innervation volume. The sophisticated Drosophila genetic toolkit enables the systematic dissection of the cellular and molecular mechanisms mediating brain circuit remodeling.

<meta charset="utf8"></head><body>Autor em destaque: Explorando a influência glial na poda sináptica dependente da experiência durante períodos críticos

We describe here methods for inducing and analyzing olfactory experience-dependent remodeling of antennal lobe synaptic glomeruli in the Drosophila juvenile brain.

Remodelação dependente da experiência da conectividade sináptica do neurônio sensorial olfativo do cérebro juvenil em um período crítico no início da vida

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Remodelação dependente da experiência da conectividade sináptica do neurônio sensorial olfativo do cérebro juvenil em um período crítico no início da vida

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