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. הידרה תרבות ומדידה של תגובת ההאכלה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשמור על פוליפים מפלצת בתרבות על ידי האכלה להם מדי יום עם הארטמיה ולשמור אותם במדיום (חיץ 1 מ&quot;מ טריס-HCl, pH 7.6, 1 מ&quot;מ NaCl, 1 מ&quot;מ CaCl 2, 0.1 מ&quot;מ KCl, ו0.1 מ&quot;מ MgSO 4) הכלולים ב קערת זכוכית על 18 מעלות צלזיוס תחת מחזורים כהים שעות אור-12 12 שעות כפי שתוארה קודם לכן 12. </li><li style=";text-align:right;direction:rtl"> למדידת תגובת ההאכלה, להעביר את אחד להבשיל פוליפ מפלצת שיש 5 עד 6 זרועות ולבודדות של צלחת 24 גם. הסר את מדיום השייר מהבאר על ידי הטיית אותו, ולאחר מכן להוסיף באופן מיידי 500 μl של מדיום חדש. </li><li style=";text-align:right;direction:rtl"> הכן פתרון גלוטתיון 9 מיקרומטר במדיום מפלצת. מאז פתרון גלוטתיון הוא נוטה לחמצון, תמיד להשתמש בפתרון גלוטתיון מוכן טרי עבור כל ניסוי. </li><li style=";text-align:right;direction:rtl"> העבר את הצלחת לפלטפורמת ההדמיה של מיקרוסקופ שיש הפרשות להקלטת תמונה. השתמש ברקע כהה באופן שo ההתנהגותפוליפ מפלצת F ניתן הדמיה בבירור על הרקע המנוגד. </li><li style=";text-align:right;direction:rtl"> החדר המשמש לצפייה והדמית ההתנהגות חופשית מפלצת מאורות בעוצמות משתנות, זרמי אוויר ורעש. הפרעות כאלה עלולות לגרום גם פוליפ המפלצת להראות התכווצות של זרועות - גם בהיעדרו של גלוטתיון. </li><li style=";text-align:right;direction:rtl"> לאפשר פוליפ להירגע במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> ודא שפוליפ ממוקם לאורך האזור המרכזי של הבאר, כך שההתנהגות ניתן הדמיה ברורה. אם פוליפ הוא בקצה של הבאר, להביא אותה למרכז על ידי שטיפה הבינוני באמצעות פיפטה ושוב לאפשר לו להירגע. </li><li style=";text-align:right;direction:rtl"> ללכוד תמונה של מפלצת במצב רגוע. זה יהיה תצפית נקודת אפס-הזמן. </li><li style=";text-align:right;direction:rtl"> להוסיף במהירות 9 מיקרומטר פתרון גלוטתיון להגיע לריכוז סופי של 3 מיקרומטר בבאר. בהתאם לצורך של הניסוי והתגובה מוצג על ידי מפלצת, לבדוק מגוון של ריכוזים שונים של גלוטתיון וChoose הריכוז המתאים הנדרש. </li><li style=";text-align:right;direction:rtl"> להפעיל את הטיימר מייד לאחר הוספת פתרון גלוטתיון וללכוד תמונות אחרי כל שניות 15-30 ל4-5 דקות. אין לשנות את הגדרות ההגדלה במהלך ההדמיה הזמן לשגות. </li><li style=";text-align:right;direction:rtl"> מוסיף את פתרון גלוטתיון בעדינות ובזרימה אחידה כך שעמדתו של בעל החיים ובתהיה מוטרדת מינימאלית בשדה הראייה של המיקרוסקופ. עם זאת, אם פוליפ נע בהרחבה לאחר הוספת פתרון גלוטתיון, להזיז את הצלחת בעדינות רבה כדי להביא את פוליפ בשדה הראייה ללכידת תמונה. </li><li style=";text-align:right;direction:rtl"> בניסוי השליטה, להשתמש במדיום חסר גלוטתיון תוך שמירה על כל הפרמטרים האחרים זהים. </li><li style=";text-align:right;direction:rtl"> הקפד לבצע את כל שלבי ניסוי מעל במחצית הראשונה של היום - לפני 13:00 להימנע ההשפעה האפשרית של קצב היממה על מידת היענות האכלה. </li><li style=";text-align:right;direction:rtl"> פתח כל אחת מהתמונות שנתפסו באמצעות תכנית GNU תמונת מניפולציה (GIMP). </li&gt; <li style=";text-align:right;direction:rtl"> השתמש בכלי &quot;מדוד&quot; זמין מתפריט&gt; כלים&gt; למדוד כדי לקבוע את המרחק בין קצה קודקוד של כל אחד מהזרועות וhypostome. אם פתיחת הפה הוא ציין בכל אחת מהתמונות, לקבוע את המרחק בין המרכז של הפה נפתח וסוף הפסגה של הזרוע. עיין במרחק זה כמו התפשטות הזרוע. </li><li style=";text-align:right;direction:rtl"> לחשב את התפשטות הזרוע הממוצעת לכל פוליפ לפני ואחרי חשיפת גלוטתיון. לחשב את היחס של התפשטות זרוע ממוצעת בנקודת אפס זמן לזה שבכל אחד מנקודתי הזמן שלאחר מכן. יחס זה נקרא התפשטות זרוע היחסית. </li><li style=";text-align:right;direction:rtl"> חזור על מדידות לפחות 20 פוליפים. </li></ol><img alt="משוואת 1" fo:content-width="3.5in" src="/files/ftp_upload/52178/52178eq1.jpg" width="400" /> <img alt="משוואה 2" fo:content-width="4in" src="/files/ftp_upload/52178/52178eq2.jpg" width="450" /> 2. שיטת אימות באמצעותדגם רעב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לרעב, להעביר כמה פוליפים הידרה לקערת זכוכית נפרדת ולא להאכיל אותם במשך 5 ימים. להאכיל את קבוצת הביקורת של כמה פוליפים יומיים עם הארטמיה בקערה בגודל דומה. לשנות את המדיום משני ניסויי קערות יומיים, כדי למנוע צמיחת פטרייה במדיום. </li><li style=";text-align:right;direction:rtl"> ביום של הניסוי, להאכיל את קבוצת הביקורת של מפלצת עם הארטמיה עבור שעה 1 ולהשתמש בהידרה אלה לניסויים הבאים לאחר הסרת כל הארטמיה לא נאכלה ומת מהמדיום. </li><li style=";text-align:right;direction:rtl"> למדוד את תגובת ההאכלה של המפלצת הרעבה בהשוואה למפלצת מקבוצת ביקורת בשיטה שתוארה קודם לכן בשלב 1. כדי למנוע כל הטיה בשל זמן של התבוננות, להחליף את המידות של כל אחד מרעב ולשלוט פוליפים מפלצת. </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.

התנהגות אכילה בהידרה מייצגת את אחת ממערכות chemosensory האבות ביותר בmetazoans. למרות הנוכחות של GSH בנוזל הסרטנים שוחרר לאחר לכידת טרף בסיוע nematocyst זוהתה מזמן 1, לא חלבון GSHR ולא את גן הקידוד המשוער / s מתאפיינת ממפלצת עד כה. ניסיונות מעטים שנעשו לאפיין GSH מחייב חלבונים במפלצת...

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.

גלוטתיון גורם מפלצת להציג סלסול של זרועות לכיוון הפה לצורך שוטף את הטרף. סלסול כזה של זרועות מביא קצות הפסגה של זרועות קרובה יותר לhypostome. התוצאה היא ההפחתה במרווח הזרוע, או את המרחק ליניארי בין סוף הפסגה של הזרוע וhypostome (איור 1). ההתפשטות היחסית הזרוע, או היחס ש?...

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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.

מדידת תגובת האכלה מושרה גלוטתיון בהידרה

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

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11.3K Views.  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.

Video: 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.

הדמיה גישות הערכות של רעילות לחץ חימצוני באמצעות חיישנים Fluorogenic מקודד גנטית

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

Cancer Research

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.

שילוב של צריכת המזון כמותיים מבחני והפעלת בכפייה נוירונים ללמוד תיאבון בשנת דרוזופילה

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

Behavior

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 ...

Biochemistry

בדיקת תפוקה גבוהה למדידת Caenorhabditis elegans צריכת מזון ותוחלת חיים

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.

מחבר זרקור: חקר הקשר בין ליפוטוקסיות ו- HFpEF 

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

כימות צריכת המזון ב- Caenorhabditis elegans על ידי מדידת פינוי חיידקים

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

מדידת GSH/GSSG – בדיקת OPA מעודנת למחקר נוגד חמצון

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.

מחבר זרקור: כימות חדשני של תיול וזיהוי סמנים ביולוגיים לחקר עקה חמצונית 

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.

כימות מהיר של צורות מחומצנות ומופחתות של גלוטתיון באמצעות אורתו-פתאלדהיד בתאי יונקים בתרבית במבחנה

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.

דור ותחזוקה לטווח ארוך של עצבים ללא<em&gt; הידרה</em

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

מדידת תגובת האכלה מושרה גלוטתיון בהידרה

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הדמיה גישות הערכות של רעילות לחץ חימצוני באמצעות חיישנים Fluorogenic מקודד גנטית

שילוב של צריכת המזון כמותיים מבחני והפעלת בכפייה נוירונים ללמוד תיאבון בשנת דרוזופילה

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

כימות צריכת המזון ב- Caenorhabditis elegans על ידי מדידת פינוי חיידקים

כימות צריכת המזון ב- Caenorhabditis elegans על ידי מדידת פינוי חיידקים

כימות צריכת המזון ב- Caenorhabditis elegans על ידי מדידת פינוי חיידקים

כימות מהיר של צורות מחומצנות ומופחתות של גלוטתיון באמצעות אורתו-פתאלדהיד בתאי יונקים בתרבית במבחנה

כימות מהיר של צורות מחומצנות ומופחתות של גלוטתיון באמצעות אורתו-פתאלדהיד בתאי יונקים בתרבית במבחנה

כימות מהיר של צורות מחומצנות ומופחתות של גלוטתיון באמצעות אורתו-פתאלדהיד בתאי יונקים בתרבית במבחנה

דור ותחזוקה לטווח ארוך של עצבים ללא