Agradecemos Joachim Erber por nos apresentar o paradigma de aprendizagem tátil em abelhas. Este trabalho foi apoiado pelo Cluster de Excelência 277 CITEC, financiado no âmbito da Iniciativa de Excelência alemão.

1. Preparando as abelhas <ol><li> Forrageiras néctar ou pólen são capturados no campo a partir de um alimentador de sacarose ou diretamente a partir da entrada colméia ao retornar de uma viagem de forrageamento. Cada única abelha é capturado dentro de um frasco de vidro, que é fechada com um tampão de espuma e levado imediatamente para o laboratório para posterior manipulação. </li><li> No laboratório, as abelhas capturados são brevemente arrefecida no frigorífico a 4 ° C até se mostrar os primeiros sinais de imobilidade. </li><li> Cada abelha única imobilizada é montado em um pequeno tubo de metal com fita adesiva entre a cabeça e no tórax e sobre o abdómen. Cuidados devem ser tomados para que a tromba e antenas são livremente móvel. </li><li> Pinte os olhos compostos e ocelos da abelha fixo com tinta branca (por exemplo, livre de solvente Tipp-Ex) para ocluir visão. </li><li> Adicionar uma pequena gota de cera derretida por trás da cabeça da abelha para corrigi-lo para a fita entre a cabeça eo tórax para evitar movimentos da cabeça durante as gravações. </li><li> Marcar cada abelha com um número único na fita para facilitar a identificação e colocar o tubo com a abelha fixo em uma atmosfera húmida para evitar a desidratação. </li><li> Alimentar cada abelha único durante 5 segundos, com gotículas de uma solução de sacarose a 30% apresentaram uma seringa e que todas as abelhas recuperar durante 30 min antes de iniciar o protocolo de condicionamento táctil. </li></ol> 2. Condicionado tátil <ol><li> Antes de condicionamento, cada abelha individual tem de ser testado para a resposta da extensão da probóscide (PER) a um estímulo de 30% de sacarose aplicada às antenas. Deste modo, a ponta do nariz tem de atravessar uma linha virtual entre as mandíbulas abertas. Descartar todas as abelhas que não respondem com um PER para o estímulo de sacarose. </li><li> Para o condicionamento táctil utilizar um cubo de metal (por exemplo, 3 x 5 mm) com uma superfície lisa ou um padrão gravado, por exemplo, horizontal ou ranhuras verticais que formam uma grade com comprimento de onda de 150 um, como o estímulo condicionado (CS). Fou o estímulo não condicionado (EUA) utilizar uma solução de sacarose a 30% (açúcar doméstico, diluído em água). </li><li> O cubo de latão (CS) é colocado sobre um suporte de um micromanipulador (por exemplo, Märzhäuser MM33) para garantir o posicionamento exacto durante o processo de condicionamento. Os EUA é apresentada ao abelha com uma seringa cheia com uma solução de sacarose a 30%. </li><li> O procedimento de condicionamento consiste em cinco pares de estímulo táctil (CS) e uma solução de sacarose (EUA), com um intervalo inter-ensaio (ITI) de 5 min. Colocar uma única abelha em frente do micromanipulador com o estímulo táctil montado (CS). Posicionar o CS lentamente, de tal modo que a superfície do estímulo táctil é paralela à cabeça da abelha (Figura 1 A e B). A distância entre o animal e o objeto tátil deve estar no intervalo de o raio da antena de trabalho da abelha testado, isto é, a abelha deve ser capaz de verificar o estímulo táctil numa posição confortável wantenas tanto de ordem i. No exemplo mostrado na Figura 1, a distância era de 3 mm. Deixe a abelha digitalizar o estímulo tátil (CS) por 5 seg. Após o primeiro 3 seg, apresentar uma gotícula da sacarose a 30% (EUA) com uma solução de uma seringa sob a tromba. Utilize a ponta da seringa para levantar suavemente as tromba. Estimulação de sacarose sob a tromba vai provocar o incondicionado PER 15. Permitir que a abelha a lamber a recompensa de sacarose. Usar um cronometro com um sinal de alarme para manter os intervalos exactos de tempo durante o condicionamento. </li><li> O PER condicionado é usada como uma medida para o sucesso da aprendizagem de uma abelha. Após os sorteios primeiro recompensados, as abelhas começam a responder a CS apresentação por extensão de seus probóscides, indicando que eles esperam a recompensa futura. A tromba totalmente estendida observados a qualquer momento durante a janela de tempo de 3 segundos de apresentação do estímulo tátil e antes da apresentação de sacarose é classificada como uma resposta positiva. Nenhuma resposta é contado negativo. O occurrence do PER tem de ser observado pelo experimentador. O percentual de abelhas que mostram o PER durante a apresentação CS é traçado para cada tentativa. </li></ol> 3. Gravações cinemáticas <ol><li> Movimentos antenais de uma única abelha aproveitado são gravados com uma câmera de vídeo digital com uma lente macro adequado (por exemplo Basler A602f-2 equipado com TechSpec VZM 200, operado a 50 fps através de uma conexão de fio de fogo). A câmara é posicionada acima do animal numa vista de cima para baixo (Figura 1 A). Na Figura 1, a resolução espacial das gravações é de 0,02 mm / pixel. </li><li> Calibrar a câmera gravando imagens únicas de um tabuleiro de xadrez 10 mm x 10 com uma aresta de comprimento 1 mm diferentes orientações no âmbito do objectivo da câmara. A calibração pode ser feita com a caixa de ferramentas da câmara de calibração Matlab 16. </li><li> Coloque uma única abelha fixa abaixo da lente da câmera. Apresentar o estímulo táctil, fixada a um micromanipulador, Para o animal. Proceder do mesmo modo como descrito para o condicionamento táctil, e gravar o movimento da antena enquanto a abelha é digitalizar o objeto. É importante que o intervalo de trabalho completo e antenal do estímulo táctil são visíveis. As escolhas de padrões de estímulo testado e números de ensaios dependem do projeto experimental. </li></ol> 4. Análise de Dados <ol><li> O cálculo da imagem de fundo é feito em Matlab. Primeiro, o valor médio tons de cinza ao longo do tempo tem que ser calculado para cada pixel. Objetos estáticos, como a cabeça fixa da abelha e do estímulo tátil, irá constituir o fundo da imagem. Os objectos em movimento, como a duas antenas, não irá fazer parte do fundo. </li><li> Cada quadro do vídeo gravado deve ser carregada sequencialmente, e a diferença entre a imagem actual e a imagem de fundo tem de ser calculada. O resultado desta subtração enfatiza as partes da imagem que está se movendo de um quadro para outro. Ideally, as antenas da abelha são as únicas áreas de valores diferentes de zero (Figura 1C). </li><li> Para processamento adicional, as duas maiores áreas com valores diferentes de zero de pixel são assumidos como sendo as antenas. Para ambas as antenas de uma máscara binária deve ser gerado para cada pixel contendo um valor de 1, se o pixel pertence à antena e 0 valor de outra forma. Esta máscara serve como uma base para a localização das pontas antenais mais tarde. Para ganhar uma máscara preliminar para a totalidade da imagem do cinzento-valor de cada pixel da imagem de diferença é comparada com um limiar pré-definido. Desde que cuidar de ruído ainda este limiar é escolhido para ser bastante baixa. A máscara de preliminar, então ainda tem dois tipos de erros: em primeiro lugar, devido a regiões da imagem de ruído pequenas ainda fazem parte da máscara. Em segundo lugar, as superfícies que pertencem à antena pode não ser necessariamente totalmente ligado. A última ocorre principalmente quando o fundo tem aproximadamente a mesma que a luminescência da antena. Para eliminar esses artefatos, padrãooperação de processamento de imagem morfológica são aplicadas, ou seja, uma combinação de imagem erosão e dilatação, contando com as funções padrão do Matlab imerode e imdilate respectivamente (ver 17 pp 158-205 para mais explicações). Após o processo de suavização das hipóteses antenas estão ainda contidos no interior da mesma máscara. Assim, como um próximo passo a máscara binária é agrupado para áreas disjuntas usando o padrão Matlab função bwlabel (veja 17 pp 40-48 para mais detalhes sobre o algoritmo de segmentação de imagem). </li><li> O número de pixels por cluster é contado e os dois maiores grupos são selecionados. O centro de gravidade é calculada para distinguir entre a antena do lado esquerdo e direito (Figura 1D). </li><li> A ponta da antena para cada antena pode ser definida como o pixel de um agrupamento com o valor mais elevado na direcção proximal para distal (ver Figura 1D). </li></ol>

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Neurosci. 4, 46 (2010).</li><li>Scheiner, R., Barnert, M., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+water+and+sucrose+responsiveness+during+the+foraging+season+affects+proboscis+extension+learning+in+honey+bees.">Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees.</a> Apidologie. 34, 67-72 (2003).</li><li>Mujagic, S., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance,+discrimination+and+proboscis+responses+of+honey+bees+(Apis+mellifera+L.)+in+the+field+and+the+laboratory.">Sucrose acceptance, discrimination and proboscis responses of honey bees (Apis mellifera L.) in the field and the laboratory.</a> J. Comp. Physiol. 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A. 181, 355-365 (1997).</li><li>Lambin, M., D&#233;glise, P., Gauthier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+movements+as+indicators+of+odor+detection+by+worker+honeybees.">Antennal movements as indicators of odor detection by worker honeybees.</a> Apidologie. 36, 119-126 (2005).</li></ol>

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Preparação das abelhas: Coleta e fixação das abelhas deve ser feito rapidamente, a fim de manter o nível de estresse do baixo animal. O stress tem um efeito sobre a capacidade de resposta PER-e, portanto, pode ter um efeito indirecto sobre o desempenho da aprendizagem em abelhas 19, 20. O nível de tensão pode ser reduzida ao colocar os frascos de vidro com as abelhas directamente em gelo imediatamente após a colheita para imobilizá-los rapidamente. Tem que ser tido em conta que as ab...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Forrageiras pólen, Apis mellifera L. </td><td></td><td></td><td> Colméias privadas </td></tr><tr><td> Frascos de vidro (22 x 45 mm) </td><td> Carl Roth GmbH &amp; Co </td><td> X655.1 </td><td></td></tr><tr><td> Tubos de bronze, 10 x 30 mm </td><td></td><td></td><td> Auto-produzido </td></tr><tr><td> Cubo de latão, 3 x 5 mm </td><td></td><td></td><td> Auto-produzido </td></tr><tr><td> Tipp-Ex (sem solventes) </td><td> Tipp-Ex GmbH &amp; Co KG </td><td></td><td></td></tr><tr><td> Solução de sacarose (30%) </td><td></td><td></td><td> Suger casa diluído em água </td></tr><tr><td> Etanol (70%) </td><td> Carl Roth GmbH &amp; Co </td><td> 9065,3 &lt;/ Td&gt; <td></td></tr><tr><td> Seringa (1 ml) e agulha (0,6 x 25) </td><td> Carl Roth GmbH &amp; Co </td><td> 59,1 </td><td></td></tr><tr><td> Cronômetro </td><td> Carl Roth GmbH &amp; Co </td><td> L423.1 </td><td></td></tr><tr><td> Micromanipulador </td><td> Märzhäuser MM33 </td><td> 00-42-101-0000 </td><td></td></tr><tr><td> Câmera de vídeo digital </td><td> Basler A602f-2 </td><td></td><td></td></tr><tr><td> Lente macro para a câmera </td><td> TechSpec VZM 200 </td><td></td><td></td></tr><tr><td> Matlab R2009b </td><td> The MathWorks </td><td></td><td></td></tr></tbody></table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

As abelhas melíferas (Apis mellifera L.) são insetos eussociais e bem conhecidos por sua complexa divisão do trabalho e capacidade de aprendizagem associativa 1, 2. As abelhas operárias passar a primeira metade de sua vida dentro da colméia escuro, onde estão as larvas de enfermagem ou a construção dos favos hexagonal regular de alimentos (por exemplo, pólen ou néctar) e prole 3. As antenas são extraordinários antenas multissensoriais e desempenham um papel fundamental em diversas funções táteis mediadas 4, incluindo a construção colméia 5 e 6 de reconhecimento de padrões. Mais tarde na vida, cada única abelha deixa a colméia para procurar alimentos. Em seguida, uma abelha tem que aprender a discriminar fontes de alimento rentáveis, memorizar a sua localização, e comunicar a seus companheiros de ninho 7. As abelhas utilizam diferentes sinais florais como cores ou odores 7, 8, mas também pistas tácteis da superfície pétala 9 para formar memórias multissensoriais da fonte de alimentação. Em laboratório conditions, as abelhas podem ser treinadas em um paradigma de aprendizagem apetitiva discriminar características do objeto tátil, como bordas ou sulcos com suas antenas 10, 11, 12, 13. Este paradigma da aprendizagem é estreitamente relacionado com o condicionamento clássico olfactory extensão da resposta tromba (PER) em abelhas encapsuladas 14. A vantagem do modelo de aprendizagem tátil no laboratório é a possibilidade de combinar as experiências comportamentais na aprendizagem com várias medições fisiológicas, incluindo a análise do padrão de movimento da antena.

No experimento seguinte estudou como o aprendizado afeta o comportamento tátil amostragem antenais de abelhas. Para isso, é monitorado o movimento da ponta da antena de abelhas naive e condicionado antes e durante a apresentação de um estímulo táctil. Em primeiro lugar, o movimento espontâneo antenal de um grupo de forrageadoras pólen (N = 42) foi registado durante 1 min. Metade das abelhas (N = 21) foi, em seguida, condicionado por emparelhamento cinco vezes um estímulo táctil com...

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

Neste protocolo, mostramos como condição aproveitado abelhas aos estímulos táteis e introduzir uma técnica de captura de movimento 2D para analisar a cinemática de multa escala padrão de amostragem antenal.

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 ...

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

10.5K visualizações.  Bielefeld University. Neste protocolo, mostramos como condição aproveitado abelhas aos estímulos táteis e introduzir uma técnica de captura de movimento 2D para analisar a cinemática de multa escala padrão de amostragem antenal.

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

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Monitoramento Eye Movement of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Neurociência

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Farmacologia Comportamental no condicionamento clássico da Resposta Extensão Proboscis em Abelhas ( Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Análise Sholl automatizado de Morfologia Neuronal Digitized em múltiplas escalas

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and focused on psychological aspects. Until now, objective observation of neurophysiological parameters has not been carried out - undoubtedly because the technical and methodological means have not been available -, investigations into the neurophysiological effects of weightlessness are in their infancy (Schneider et al. 2008).
While imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) would be hardly applicable in space, the non-invasive near-infrared spectroscopy (NIRS) technique represents a method of mapping hemodynamic processes in the brain in real time that is both relatively inexpensive and that can be employed even under extreme conditions. The combination with electroencephalography (EEG) opens up the possibility of following the electrocortical processes under changing gravity conditions with a finer temporal resolution as well as with deeper localization, for instance with electrotomography (LORETA).
Previous studies showed an increase of beta frequency activity under normal gravity conditions and a decrease under weightlessness conditions during a parabolic flight (Schneider et al. 2008a+b). Tilt studies revealed different changes in brain function, which let suggest, that changes in parabolic flight might reflect emotional processes rather than hemodynamic changes. However, it is still unclear whether these are effects of changed gravity or hemodynamic changes within the brain. Combining EEG/LORETA and NIRS should for the first time make it possible to map the effect of weightlessness and reduced gravity on both hemodynamic and electrophysiological processes in the brain. Initially, this is to be done as part of a feasibility study during a parabolic flight. Afterwards, it is also planned to use both techniques during medium- and long-term space flight.
It can be assumed that the long-term redistribution of the blood volume and the associated increase in the supply of oxygen to the brain will lead to changes in the central nervous system that are also responsible for anaemic processes, and which can in turn reduce performance (De Santo et al. 2005), which means that they could be crucial for the success and safety of a mission (Genik et al. 2005, Ellis 2000).
Depending on these results, it will be necessary to develop and employ extensive countermeasures. Initial results for the MARS500 study suggest that, in addition to their significance in the context of the cardiovascular and locomotor systems, sport and physical activity can play a part in improving neurocognitive parameters. Before this can be fully established, however, it seems necessary to learn more about the influence of changing gravity conditions on neurophysiological processes and associated neurocognitive impairment.

The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. 
A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.

Coerência entre a função cerebral cortical e Desempenho neurocognitiva durante condições de gravidade mudou

Previous studies of cognitive, mental and/or motor processes during short-, medium- and long-term weightlessness have only been descriptive in nature, and ...

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 ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Medidas eletrofisiológicas e Análise de nocicepção em bebês humanos

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Determinação rápida do limiar térmico nociceptivo em ratos diabéticos

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi mediado por Gene Knockdown duplo e Mensuração Percepção gustativa em abelhas do mel (<em&gt; Apis mellifera</em&gt;)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Manipulação neurofarmacologia de contido e vôo livre abelhas do mel,<em&gt; Apis mellifera</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

O uso do condicionamento de rastreamento Eyeblink clássico para avaliar disfunção Hippocampal em um modelo do rato de transtornos do espectro do álcool Fetal

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...