Name: measuring glutathione-induced feeding response in hydra
Uploaded: 2014-11-16T15:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Measuring Glutathione-induced Feeding Response in Hydra at JoVE.com

Authors are thankful to K. P. Madhu, Nita Beliappa and staff of the Media Centre of Indian Institute of Science Education and Research, Pune for their help in the video production. The work was supported by funding under the Centre of Excellence program of Department of Biotechnology, Government of India to SG and postdoctoral fellowship by Department of Science and Technology, Government of India to RK.

1. Hydra Cultura e medição da resposta Alimentação <ol><li> Manter pólipos hidra em cultura, alimentando-os com diária de artemia e mantendo-os num meio (tampão de 1 mM Tris-HCl, pH 7,6, NaCl a 1 mM, CaCl2 1 mM, KCl 0,1 mM, e 0,1 mM de MgSO4) contida em um tigela de vidro a 18 ° C sob ciclos escuros hr de luz 12 12 h, como descrito anteriormente 12. </li><li> Para a medição da resposta de alimentação, transferir um pólipo amadurecer hidra tendo 5 a 6 tentáculos para um único poço de uma placa de 24 poços. Retirar o meio residual do bem, inclinando-lo, e em seguida, adicione imediatamente 500 mL de meio fresco. </li><li> Preparar a solução de glutationa 9 uM em meio de hidra. Uma vez que a solução de glutationa é propenso a oxidação, usar sempre a glutationa solução preparada de fresco para cada experiência. </li><li> Transferir a placa para a plataforma de imagem de um microscópio tendo disposições para a gravação da imagem. Usar um fundo escuro de tal modo que o comportamentof pólipo hidra pode ser claramente fotografada contra o fundo contrastante. </li><li> A sala utilizada para a observação e imagem do comportamento de hidra livre de luzes de intensidade flutuante, correntes de ar e de ruído. Tais perturbações podem também fazer com que o pólipo hidra para mostrar a contracção dos tentáculos - mesmo na ausência de glutationa. </li><li> Permitir que o pólipo para relaxar por 5 min. </li><li> Certifique-se de que o pólipo está localizado ao longo da região central da cavidade de tal modo que o comportamento pode ser trabalhada de forma clara. Se o pólipo está na borda do poço, trazê-la para o centro, através de lavagem do meio utilizando uma pipeta e novamente permitir que ele relaxe. </li><li> Capturar uma imagem da hidra no estado relaxado. Este será o ponto de observação de tempo zero. </li><li> Rapidamente adicionar 9 uM solução glutationa para alcançar uma concentração final de 3 uM no poço. Dependendo da finalidade da experiência e a resposta mostrada por hidra, testar uma gama de diferentes concentrações de glutationa e Chooif a concentração adequada e necessária. </li><li> Inicie o cronômetro imediatamente após a adição de solução de glutationa e capturar imagens após cada 15-30 seg para 4-5 min. Não altere as configurações de ampliação durante a imagiologia de lapso de tempo. </li><li> Adicionar a solução de glutationa suavemente e com um fluxo uniforme de tal modo que a posição do animal no poço seria minimamente perturbado no campo de visão do microscópio. No entanto, se o pólipo se move extensivamente após a adição de solução de glutationa, mover a placa muito suavemente para trazer o pólipo no campo de visão para a captura de imagem. </li><li> No experimento de controle, use meio sem glutationa, mantendo todos os outros parâmetros idênticos. </li><li> Certifique-se de executar todas as etapas experimentais acima durante a primeira metade do dia - antes de 13:00 para evitar o possível efeito do ritmo circadiano sobre a extensão da resposta de alimentação. </li><li> Abra cada uma das imagens capturadas usando o GNU Image Manipulation Program (GIMP). </lEu&gt; <li> Use a ferramenta &quot;Medida&quot;, disponível a partir de Menu&gt; Ferramentas&gt; Medida para determinar a distância entre a extremidade apical de cada um dos tentáculos e hypostome. Se a abertura boca é observado em qualquer uma das imagens, determinar a distância entre o centro da boca aberta e a extremidade apical do tentáculo. Consulte a esta distância como a propagação tentáculo. </li><li> Calcule o spread médio tentáculo para cada pólipo antes e depois da exposição a glutationa. Calcular a proporção de spread médio tentáculo no ponto de tempo zero para que em cada um dos pontos de tempo subsequentes. Esta relação será chamado propagação tentáculo relativa. </li><li> Repita as medições por pelo menos 20 pólipos. </li></ol><img alt="Equação 1" fo:content-width="3.5in" src="/files/ftp_upload/52178/52178eq1.jpg" width="400" /> <img alt="Equação 2" fo:content-width="4in" src="/files/ftp_upload/52178/52178eq2.jpg" width="450" /> 2. método de validação usando oA fome Modelo <ol><li> Para a fome, transferir alguns pólipos Hydra para uma tigela de vidro separada e não alimentá-los durante 5 dias. Alimente o grupo de alguns pólipos controle diário com artemia em uma tigela de tamanho similar. Mudar o meio de tanto experimental taças diariamente para evitar crescimento de fungos no meio. </li><li> No dia do experimento, alimentar o grupo de controle hidra com artemia por 1 hr e usá-los hidra para os experimentos posteriores após a remoção de todos artemia não consumidos e mortos a partir do meio. </li><li> Meça a resposta de alimentação da hidra fome em comparação com a hidra do grupo de controle pelo método descrito anteriormente na etapa 1. Para evitar qualquer viés devido ao tempo de observação, alternar as medidas de cada da fome e controlar pólipos hidra. </li></ol>

<ol>
	<li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+control+of+the+specific+feeding+reactions+of+hydra.">Glutathione control of the specific feeding reactions of hydra.</a> Ann. Ny. Acad. Sci. 62, 209-228 (1955).</li><li>Beckmann, A., Ozbek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nematocyst:+a+molecular+map+of+the+Cnidarian+stinging+organelle.">The Nematocyst: a molecular map of the Cnidarian stinging organelle.</a> Int. J. Dev. Biol. 56, 577-582 (2012).</li><li>Venturini, G., Carolei, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+receptors+in+Hydra.+Pharmacological+and+biochemical+observations.">Dopaminergic receptors in Hydra. Pharmacological and biochemical observations.</a> Comp. Biochem. Phys. C. 102, 39-43 (1992).</li><li>Kass-Simon, G., Scappaticci, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+and+GABAnergic+control+in+the+tentacle+effector+systems+of+Hydra+vulgaris.">Glutamatergic and GABAnergic control in the tentacle effector systems of Hydra vulgaris.</a> Hydrobiologia. 530-531, 67-71 (2004).</li><li>Pierobon, P., Tino, A., Minei, R., Marino, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+roles+of+GABA+and+glycine+in+the+modulation+of+chemosensory+responses+in+Hydra+vulgaris+(Cnidaria,+Hydrozoa).">Different roles of GABA and glycine in the modulation of chemosensory responses in Hydra vulgaris (Cnidaria, Hydrozoa).</a> Hydrobiology. 178, 59-66 (2004).</li><li>Pierobon, P., Sogliano, C., Minei, R., Tino, A., Porcu, P., Marino, G., Tortiglione, C., Concas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putative+NMDA+receptors+in+Hydra:+a+biochemical+and+functional+study.">Putative NMDA receptors in Hydra: a biochemical and functional study.</a> Eur. J. Neurosci. 20, 2598-2604 (2004).</li><li>Alzugaray, M. E., Adami, M. L., Diambra, L. A., Hernandez-Martinez, S., Damborenea, C., Noriega, F. G., Ronderos, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allatotropin:+An+ancestral+myotropic+neuropeptide+involved+in+feeding.">Allatotropin: An ancestral myotropic neuropeptide involved in feeding.</a> PLoS ONE. 8, (2013).</li><li>Bellis, S. L., Laux, D. C., Rhoads, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+Hydra+glutathione+binding+proteins.">Affinity purification of Hydra glutathione binding proteins.</a> FEBS Lett. 354, 320-324 (1994).</li><li>Lenhoff, H. M., Shaw, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+of+the+GSH+receptor+in+Hydra+and+its+evolutionary+significance.">The discovery of the GSH receptor in Hydra and its evolutionary significance.</a> Glutathione in the Nervous System. , 25-43 (1998).</li><li>Venturini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydra+GSH+receptor.+Pharmacological+and+radioligand+binding+studies.">The hydra GSH receptor. Pharmacological and radioligand binding studies.</a> Comp. Biochem. Phys. C. 87, 321-324 (1987).</li><li>Reddy, P. C., Barve, A., Ghaskadbi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+and+phylogenetic+characterization+of+common+hydra+from+India.">Description and phylogenetic characterization of common hydra from India.</a> Curr. Sci. 101, 736-738 (2011).</li><li>Horibata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+catabolic+pathway+of+glycosphingolipids+in+a+hydrozoan,+Hydra+magnipapillata.+Involving+endoglycoceramidase.">Unique catabolic pathway of glycosphingolipids in a hydrozoan, Hydra magnipapillata. Involving endoglycoceramidase.</a> J. Biol. Chem. 279, 33379-33389 (2004).</li><li>Koizumi, O., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rise+of+feeding+threshold+in+satiated+Hydra.">Rise of feeding threshold in satiated Hydra.</a> J. Comp. Physiol. 142, 75-80 (1981).</li><li>Bellis, S. L., Kass-Simon, G., Rhoads, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+characterization+and+detergent+solubilization+of+the+putative+glutathione+chemoreceptor+from+hydra.">Partial characterization and detergent solubilization of the putative glutathione chemoreceptor from hydra.</a> Biochemistry. 31, 9838-9843 (1992).</li><li>Morita, H., Hanai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+receptor+proteins+in+invertebrates+-+with+special+reference+to+glutathione+receptor+of+hydra.">Taste receptor proteins in invertebrates - with special reference to glutathione receptor of hydra.</a> Chem. Senses. 12, 245-250 (1987).</li><li>Colasanti, M., Venturini, G., Merante, A., Musci, G., Lauro, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+involvement+in+Hydra+vulgaris+very+primitive+olfactory-+like+system.">Nitric oxide involvement in Hydra vulgaris very primitive olfactory- like system.</a> Journal of Neurosci. 17, 493-499 (1997).</li><li>Kuhn, A., Tsiairis, C. D., Williamson, M., Kalbacher, H., Grimmelikhuijzen, C. J., Holstein, T. W., Gr&#252;nder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+homologous+subunits+form+a+high+affinity+peptide-gated+ion+channel+in+Hydra.">Three homologous subunits form a high affinity peptide-gated ion channel in Hydra.</a> J. Biol. Chem. 285, 11958-11965 (2010).</li><li>Wang, M., Yao, Y., Kuang, D., Hampson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+family+C+G-protein-coupled+receptors+by+the+tripeptide+glutathione.">Activation of family C G-protein-coupled receptors by the tripeptide glutathione.</a> J. Biol. Chem. 281, 8864-8870 (2006).</li><li>Ruggieri, R. D., Pierobon, P., Kass-Simon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaker+activity+in+hydra+is+modulated+by+glycine+receptor+ligands.">Pacemaker activity in hydra is modulated by glycine receptor ligands.</a> Comp. Biochem. Phys. C. 138, 193-202 (2004).</li><li>Ramazani, R. B., Krishnan, H. R., Bergeson, S. E., Atkinson, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+automated+movement+detection+for+the+analysis+of+behavior.">Computer automated movement detection for the analysis of behavior.</a> J. Neurosci. Meth. 162, 171-179 (2007).</li></ol>

The authors declare no competing financial interests.

Comportamento alimentar em hidra representa um dos sistemas de quimio mais ancestrais dos metazoários. Embora a presença de GSH no fluido crustáceo libertado após assistida-nematocyst captura de presa foi detectada há muito tempo 1, nem a proteína GSHR nem o gene que codifica putativo / s têm sido caracterizadas a partir da hidra até à data. Poucas tentativas têm sido feitas para caracterizar as proteínas de ligação a GSH na hidra 8, 14, 15. No entanto, a identidade destas proteínas r...

Hydra is the most primitive organism possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey1. It feeds on a variety of animals such as nematode, crustacea, insect larvae, tadpoles and newly hatched fish1. The movement of these prey organisms causes mechanosensory discharge of the stinging capsules called nematocysts from hydra, which are inserted into the prey2. GSH present in the fluid released from the injured prey subsequently activates the feeding response in hydra which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth, and consequent engulfing of the prey. Multiple molecules, such as dopamine3, glutamate4, GABA, glycine5, NMDA receptors6, and allatotropin7, have been shown to be involved in the feeding response in hydra. It has also been shown that the chemosensory response induced by GSH is modulated by the feeding status of the animal such that starved hydra exhibited enhanced feeding response1. Such an increase in the GSH sensitivity is biologically relevant since under starvation hydra need to find its prey at higher sensitivity. 
Although the feeding response induced by GSH can be clearly observed under microscope, the methods typically used for measuring the feeding response observations are non-quantitative. In most of the cases, the time during which the mouth of the hydra remains open was taken as a measure of the feeding response8,9; whereas in another case, quantitation was based on the number of hydra out of a population showing the feeding response10. However, observing the mouth opening time of the hydra polyps is cumbersome and subject to variation induced by uncontrollable parameters such as the direction of the mouth orientation during observations. Similarly, since the feeding response is a quantitative parameter, population-based approaches are subject to variations/errors caused by the opinion or observational bias of the individual observer. To circumvent these issues we have developed a method for the relative quantification of the feeding response in hydra (Hydra vulgaris Ind-Pune11) based on the distance of the apical end of the tentacle from the mouth of the hydra polyp.

<table><tbody><tr><td>Cooled Incubator</td><td>Panasonic&amp;nbsp;</td><td>MIR-254-PE</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>S8AP0&amp;nbsp;</td><td></td></tr><tr><td>Camera for the microscope</td><td>Leica&amp;nbsp;</td><td>EC3</td><td></td></tr><tr><td>Reduced glutathione</td><td>Sigma</td><td>G4251</td><td>Stored at 4 &amp;deg;C. Bring the bottle to room temperature before opening to avoid oxidation</td></tr><tr><td>Image editing program</td><td>GIMP</td><td>Version 2.8</td><td></td></tr></tbody></table>

measuring glutathione-induced feeding response in hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

A glutationa faz com que a hidra exibem ondulação dos tentáculos para a boca com a finalidade de engolindo a presa. Essa ondulação de tentáculos traz extremidades apicais dos tentáculos mais perto do hypostome. Isto resulta na redução da propagação tentáculo, ou a distância linear entre a extremidade apical do tentáculo e hipostoma (Figura 1). A propagação tentáculo parente, ou a proporção de tentáculo média espalhar antes e após a adição de glutationa, em média em vários póli...

Watch this Scientific Journal Video about Measuring Glutathione-induced Feeding Response in Hydra at JoVE.com

Measuring Glutathione-induced Feeding Response in Hydra

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Medindo alimentação resposta induzida por glutationa em Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

measuring-glutathione-induced-feeding-response-in-hydra

Research

JoVE Journal

Biology

11.3K visualizações.  India Institute of Science Education and Research, Pune. Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Vídeo: Measuring Glutathione-induced Feeding Response in Hydra

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

Abordagens para avaliação de estresse oxidativo toxicológico usando sensores Fluorogenic geneticamente codificado de imagem

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Pesquisa do câncer

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Combinando os ensaios quantitativos de ingestão de alimentos e forçosamente, ativando os neurônios para estudar o apetite em Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Comportamento

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Bioquímica

Ensaio de alto rendimento para medir a ingestão de alimentos e a expectativa de vida de Caenorhabditis elegans

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Autor em destaque: Explorando a relação entre lipotoxicidade e ICFEP 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Medição GSH/GSSG – Um ensaio OPA refinado para pesquisa antioxidante

Rapid Quantification of Oxidized and Reduced Forms of Glutathione Using Ortho -phthalaldehyde in Cultured Mammalian Cells In Vitro

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30&#181;M (R2=0.9932&#177;0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

Autor em destaque: Inovando a quantificação de tióis e a detecção de biomarcadores para pesquisa de estresse oxidativo 

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

Quantificação rápida de formas oxidadas e reduzidas de glutationa usando orto-ftalaldeído em células de mamíferos cultivadas in vitro

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Geração e Manutenção a Longo Prazo de Nerve-free<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Medindo alimentação resposta induzida por glutationa em Hydra

Resumo

Explore mais vídeos

Capítulos neste vídeo

Abordagens para avaliação de estresse oxidativo toxicológico usando sensores Fluorogenic geneticamente codificado de imagem

Combinando os ensaios quantitativos de ingestão de alimentos e forçosamente, ativando os neurônios para estudar o apetite em Drosophila

Measurement of Glutamate Uptake using Radiolabeled L-[³H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

Quantificando a ingestão de alimentos em Caenorhabditis elegans medindo a depuração bacteriana

Quantificação rápida de formas oxidadas e reduzidas de glutationa usando orto-ftalaldeído em células de mamíferos cultivadas in vitro

Quantificação rápida de formas oxidadas e reduzidas de glutationa usando orto-ftalaldeído em células de mamíferos cultivadas in vitro

Quantificação rápida de formas oxidadas e reduzidas de glutationa usando orto-ftalaldeído em células de mamíferos cultivadas in vitro

Geração e Manutenção a Longo Prazo de Nerve-free