Este trabalho é financiado pela DFG.

Bee Preparação e Back-fill <ol><li> Forrageiras pegar mel de abelha em colmeia e frio no gelo para imobilização. </li><li> Montagem em Plexiglas gravação câmaras 3. Fixar os olhos e tórax para as paredes da câmara gravando com cera baixo ponto de fusão rígido. </li><li> Puxe capilares de vidro tão comumente utilizado para eletrofisiologia e quebrar na ponta para obter um diâmetro da ponta de ca. 10μm. Cobrir a ponta do capilar com pasta de corante. Colar Dye consiste de uma mistura 10:01 de Fura-2 dextran e fixable lisina tetrametilrodamina dextran. </li><li> Para o procedimento de coloração imobilizar antenas com minúcias ou n-eicosane. Retire um pedaço de cutícula acima do cérebro e glândulas empurrar e traquéia para o lado para permitir o acesso ao corpo de cogumelo. </li><li> Injetar capilares em qualquer soma ou região axonal dos neurônios do corpo de cogumelo. Isto pode ser feito a mão livre ou através de um manipulador micro. Em seguida, restaurar parte da cutícula na cápsula de cabeça e soltar antenas. </li><li> Alimentar as abelhas com solução de sacarose 30% antes de armazená-los em um caso umidificado por pelo menos quatro horas ou durante a noite a 20 ° C. </li></ol> In vivo de imagens <ol><li> Impedir os movimentos do exemplo da abelha com um pequeno pedaço de esponja que é pressionado contra o abdômen e fixada com um clipe ou fita para a câmara de gravação. </li><li> Para impedir o movimento do cérebro resultante de bombeamento do esôfago, corte uma pequena incisão na cutícula acima do labrum e retire cuidadosamente o esôfago e seu entorno estruturas sólidas para colocá-la sob tensão, sem danificar o esôfago 4. Cubra com duas silicone componente. </li><li> Faça furos com uma agulha e inserir eletrodos de arame para a gravação de M17. </li><li> Retire parte da cutícula, traquéia e glândulas acima do cérebro. Sugar heamolymph dentro da cápsula cabeça com um pedaço de papel. </li><li> Encher a cápsula cabeça com dois silicone componente. É importante que o cérebro está completamente coberto. </li><li> Abelha lugar no palco do microscópio e coloque uma gota de água sobre a superfície do silicone para imergir o objetivo mergulho do microscópio para a gota. Concentre-se em neurônios manchado. </li></ol> Estimulação odor e gravação de sinais Olfatômetro: Nós usamos um computador controlado, dispositivo de estimulação personalizado construído ou &quot;olfatômetro&quot;, como descrito anteriormente por Galizia e Vetter 3. Odores são diluídos em um fluxo constante de ar dirigido para as antenas. Qualquer estímulo consiste de um pulso de 3s odor de ar odor 0.2ml saturada. O protocolo de estimulação pode ser definido na TILLVision software de gravação. Microscópio e Imagem set-up: Nós usamos um microscópio de fluorescência Zeiss. As imagens são gravadas a 25 ° C com uma taxa de amostragem de 5 Hz usando uma TILL-Photonics dispositivo de imagem montado no microscópio. Sinais de cálcio são registrados através de um X60, 0,9 W Olympus mergulho objetivo com um CCD Imago câmera (640X480 pixels, 4X binned em chip para 160X120 pixels). Fura-2 está animado com a luz monocromática de 340 e 380nm de comprimento de onda para medições raciométrica. Fluorescência é detectada através de um espelho dicróico 410nm e um filtro passa-440nm de comprimento. Os parâmetros para a gravação são definidas no software de gravação. Cada medição dura 10s e tempo de exposição para os dois comprimentos de onda pode ser ajustado para a intensidade de coloração em cada preparação. M17 gravação: O músculo transferidor do lábio (M17) é gravado extracelularmente para monitorar as respostas comportamentais relacionados com a aprendizagem, ou seja, os 5 PER, 6. Injetar um fio de cobre no músculo perto da partes da boca. Injetar eletrodo terra no olho. Os potenciais musculares são amplificados com um costume construir pré-amplificador, digitalizadas e armazenadas em um computador. Início do estímulo é desencadeado pela olfatômetro. Análise de Dados <ol><li> Durante o experimento: As gravações do sinal de cálcio são alimentados em um computador e armazenado. O TILLVision software de gravação permite uma inspecção inicial dos sinais, como o cálculo da relação entre os comprimentos de onda (340nm/380nm) e cálculo de deltaF (subtração da linha de base). </li><li> Processamento de imagem é realizada com o costume programas escritos em IDL. Calcular proporção de Ca 2 + sinais de 340nm e 380nm medições para cada pixel. Determine fundo de fluorescência (F) pela média de mais de frames antes do estímulo e subtrair o sinal raciométrica (deltaF). </li><li> Para definir região de interesse (ROI): Calcular média de frames durante o estímulo e aplicar um filtro passa-baixa (3X3px). Converter em escala de cinza para escala de cores falsas. Determinar a atividade neuronal em estruturas como pontos de atividade em imagens de cores falsas e definir como ROI. </li><li> Calcular dinâmica temporal das regiões ativas pela média dos pixels na ROI sobre todas as imagens sem qualquer filtragem ou correção. </li></ol> Juntamente com o dextran Fura-2 enchemos os neurônios com o corante rodamina fixable dextran (&quot;mini ruby&quot;). Ambos os corantes têm o mesmo peso molecular, que devem ser co-localizados nos neurônios. Grande campo de imagem permite apenas a resolução espacial muito limitada, portanto recomenda-se a investigar as estruturas manchado após o experimento usando microscopia confocal. <ol><li> Após as medições fisiológicas, dissecar o cérebro e se fixar. </li><li> Enxágüe em PBS e desidratar stepwise em etanol e salicilato de metila em claro. </li><li> Microscopia confocal para incorporar cérebro em salicilato de metila. </li><li> Nós usamos um microscópio Leica TCS SP2. Comprimento de onda de excitação é 543nm. </li><li> Nós escanear o cérebro com um ar ou objetiva de imersão em óleo e comparar as estruturas coradas com os dados de imagem. </li></ol> Resultados representativos: Se a coloração e preparação foi bem-sucedida, o sinal na escala de cores falsas, por exemplo, em cogumelo corpo neurônios alfa lobo extrínsecos, poderia olhar como na figura 1. A resposta comportamental (PER) pode ser calculado como freqüência de pico durante a estimulação menos freqüência pico antes do estímulo (Figura 2). As estruturas manchadas podem ser investigadas em maior detalhe o uso de um microscópio confocal (Figura 3). <img src="/files/ftp_upload/1353/figure1_new.png" alt="Figura 1" /> Figura 1: Os sinais de cálcio Representante Imaging (estimulação com heptanal). A: região fotografada do cérebro das abelhas. B: image fluorescentes Raw como pode ser visto através de um mergulho X20 objetivo no comprimento de onda de excitação 380nm, o lobo-alfa do corpo cogumelo é descrito em roxo. Corante foi injetado no rim ventro-lateral dos lobos alfa-manchar neurônios extrínsecos. C: Imagem em cor falsa derivado do sinal de média, deltaF (340/380), durante o estímulo três segundo, alfa-lobo descritas em roxo, ROI selecionada delineadas em preto. D: dinâmica temporal do sinal de cálcio em resposta à estimulação odor, sinal média da região de interesse, como indicado no C. E: dinâmica temporal do sinal de cálcio em resposta à estimulação de sacarose da ipsi-e contra-lateral da antena, respectivamente para o ROI indicado em C. <img src="/files/ftp_upload/1353/figure2_new.png" alt="" /> Figura 2: resposta M17 Representante. Após o condicionamento olfativo a abelha estende a tromba quando o odor é apresentado sem recompensa. Linha vermelha indica o estímulo do odor. Detalhe: PER da abelha. <img src="/files/ftp_upload/1353/figure3_new.png" alt="" /> Figura 3: Representante da pilha de imagens. Após a experiência de imagem do cérebro foi dissecado. O neurônio da figura 1 é digitalizado usando um microscópio confocal com uma objetiva de imersão X20 petróleo, alfa-lobo descritas em roxo.

<ol>
	<li>Joerges, J., K&#252;ttner, A., Galizia, C. G., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representations+of+odors+and+odor+mixtures+visualized+in+the+honeybee+brain.">Representations of odors and odor mixtures visualized in the honeybee brain.</a> Nature. 387 (6630), 285-288 (1997).</li><li>Rehder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+honeybee's+proboscis+reflex+by+electromyiographic+recordings.">Quantification of the honeybee's proboscis reflex by electromyiographic recordings.</a> J. Insect Physiol. 33, 501-507 (1987).</li><li>Galizia, C. G., Vetter, R. S., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Methods+for+Analyzing+Odor-Evoked+Activity+in+the+Insect+Brain.">Optical Methods for Analyzing Odor-Evoked Activity in the Insect Brain.</a> Methods in Insect Sensory Neuroscience. , 345-388 (2004).</li><li>Mauelshagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+olfactory+learning+paradigms+in+an+identified+neuron+in+the+honeybee+brain.">Neural correlates of olfactory learning paradigms in an identified neuron in the honeybee brain.</a> J Neurophysiol. 69 (2), 609-625 (1993).</li><li>Kuwabara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bildung+des+bedingten+Reflexes+von+Pavlovs+Typus+bei+der+Honigbiene+Apis+mellifica,+Hokaido+Univ.">Bildung des bedingten Reflexes von Pavlovs Typus bei der Honigbiene Apis mellifica, Hokaido Univ.</a> Zool. J. Sci. 13, 458-464 (1957).</li><li>Bitterman, M. E., Menzel, R., Fietz, A., Schafer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+of+proboscis+extension+in+honeybees+(Apis+mellifera.">Classical conditioning of proboscis extension in honeybees (Apis mellifera.</a> Journal of Comparative Psychology. 97 (2), 107-119 (1983).</li></ol>

Nesta apresentação nós já passamos por todas as etapas para investigar sinais de cálcio vivo na abelha. Nós aplicamos essa técnica para os neurônios do corpo de cogumelo, mas de imagens pode ser feito em todos os neurônios para que uma técnica de coloração pode ser estabelecida. Temos nos concentrado em neurônios na via olfativa da abelha melífera. Os experimentos são realizados em uma câmara escura. A configuração é iluminado com uma luz de onda longa que se encontra fora da faixa d...

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in vivo ca2+- imaging of mushroom body neurons during olfactory learning in the honey bee

A in vivo e semi-vivo, em preparação para a imagem latente de cálcio tem sido desenvolvido em nosso laboratório por Joerges, Küttner Galizia e mais de dez anos atrás, para medir a atividade odor evocado no lobo antenal 1. A partir de então, ele tem sido constantemente refinados e aplicados a neuropiles diferentes no cérebro das abelhas. Aqui, descrevemos a preparação atualmente usado em laboratório para medir a atividade nos neurônios do corpo usando um cogumelo acoplado dextran cálcio sensíveis corante (Fura-2). Nós retrogradamente mancha neurônios corpo de cogumelos pela injeção de corante em seus axônios ou região soma. Nós nos concentramos em reduzir a invasão, para conseguir uma preparação em que ainda é possível treinar a abelha usando PER condicionado. Somos capazes de monitorar e quantificar a resposta comportamental gravando electro-myograms do músculo que controla o PER (M17) 2. Após a experiência fisiológica das estruturas imaged são investigados em maior detalhe por microscopia confocal para tratar a identidade dos neurônios.

Watch this Scientific Journal Video about In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee at JoVE.com

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Abelhas pode ser condicionado em um paradigma de aprendizado apetitivo olfativo (PER-condicionado). Usando odores como estímulos, estabelecemos um método em que o comportamento é gravado simultaneamente imagens de cálcio é usado para medir a atividade do odor evocado nos neurônios do corpo de cogumelo<em&gt; In vivo</em&gt;.

In vivo Ca 2 + - Imagem de Neurônios Corpo Mushroom durante o aprendizado olfativo no Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

in-vivo-ca2-imaging-mushroom-body-neurons-during-olfactory-learning

Research

JoVE Journal

Biology

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

13.9K visualizações.  Freie Universität Berlin. Abelhas pode ser condicionado em um paradigma de aprendizado apetitivo olfativo (PER-condicionado). Usando odores como estímulos, estabelecemos um método em que o comportamento é gravado simultaneamente imagens de cálcio é usado para medir a atividade do odor evocado nos neurônios do corpo de cogumelo In vivo.

Vídeo: In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Imagens de cálcio de odor-provocou respostas no Drosophila Lobo antenal

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Neurociência

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

Deteção da fosfolipase C atividade no cérebro Homogenate da abelha

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

Bioquímica

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Medindo respostas fisiológicas de<em&gt; Drosophila</em&gt; Neurônios sensoriais para Lipid feromonas Usando Imagem de cálcio ao vivo

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Imunologia e Infecção

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

Gravações simultâneas de longo prazo em dois estágios de processamento neuronal em comportar abelhas

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

In Vivo funcional do cérebro Imagiologia Abordagem Baseada em cálcio Bioluminescent Indicador GFP-aequorina

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

In vivo Optical cálcio Imaging de aprendizagem induzida por plasticidade sináptica em Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Imagem Subcelular da Manipulação Neuronal de Cálcio In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

In Vivo Calcium Imaging of Taste-Induced Neural Responses in Adult Drosophila

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model organism, Drosophila melanogaster. A key strength of this methodology is its ability to record taste-induced neural responses in awake animals without the need for anesthesia. This approach employs binary expression systems (e.g., Gal4-UAS) to express the calcium indicator GCaMP in specific neurons of interest. This protocol describes a procedure in which flies expressing GCaMP are mounted with the labellum securely positioned, enabling fluorescence in the brain to be recorded at millisecond resolution under a confocal microscope while a solution is applied to the labellum, stimulating all labellar taste sensilla. The examples provided focus on calcium responses in primary gustatory receptor neurons of D. melanogaster. However, this approach can be adapted to record from other neurons of interest within the brain of Drosophilids or other insect species. This imaging method enables researchers to simultaneously record collective calcium responses from groups of gustatory neurons across the labellum, complementing electrophysiological tip recordings that quantify action potentials from individual neurons. The in vivo calcium imaging technique outlined here has been instrumental in uncovering molecular and cellular mechanisms of chemosensation, identifying unique temporal response patterns in primary taste neurons, investigating mechanisms of gustatory modulation, and exploring taste processing in downstream circuits.

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

In vivo Imagem de cálcio de respostas neurais induzidas pelo paladar em Drosophila adulta

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

Imageamento de cálcio em neurônios

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

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In Vivo Calcium Imaging Using Synaptically Localized Calcium Sensors in Drosophila melanogaster

Source: Hancock, C. E. et al., <a href="https://app.jove.com/v/60288">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster.</a>&#160;J. Vis. Exp. (2019).
This video demonstrates a method for in vivo calcium imaging using synaptically localized calcium sensors in flies. It outlines the steps for preparing the flies for microscopy, conducting olfactory conditioning, and visualizing odor-induced calcium influx.

in vivo Imagens de cálcio usando sensores de cálcio localizados sinapticamente em Drosophila melanogaster

1. Transgenic fruit flies,&#160;Drosophila melanogaster

	Cross female virgin and male flies (raised at 25 &#176;C in 60% relative humidity on a 12 h ...

In vivo Ca ^{2 +} - Imagem de Neurônios Corpo Mushroom durante o aprendizado olfativo no Honey Bee

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