Name: use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule
Uploaded: 2015-11-25T15:00:00.000+00:00
Duration: 13 min 30 s
Description: Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

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	<li>Ramsay, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+Transport+of+Water+by+the+Malpighian+Tubules+of+the+Stick+Insect,+Dixippus-Morosus+(Orthoptera,+Phasmidae).">Active Transport of Water by the Malpighian Tubules of the Stick Insect, Dixippus-Morosus (Orthoptera, Phasmidae).</a> J Exp Biol. 31, 104-113 (1954).</li><li>Dow, J. A., 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=The+malpighian+tubules+of+Drosophila+melanogaster:+a+novel+phenotype+for+studies+of+fluid+secretion+and+its+control.">The malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control.</a> J Exp Biol. 197, 421-428 (1994).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sozen, M. A., Armstrong, J. D., Yang, M., Kaiser, K., Dow, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+domains+are+specified+to+single-cell+resolution+in+a+Drosophila+epithelium.">Functional domains are specified to single-cell resolution in a Drosophila epithelium.</a> Proc Natl Acad Sci U S A. 94, 5207-5212 (1997).</li><li>Rosay, P., 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=Cell-type+specific+calcium+signalling+in+a+Drosophila+epithelium.">Cell-type specific calcium signalling in a Drosophila epithelium.</a> J Cell Sci. 110 (15), 1683-1692 (1997).</li><li>Dow, J. T., Davies, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+physiology+and+functional+genomics+of+epithelial+function+in+a+genetic+model+organism.">Integrative physiology and functional genomics of epithelial function in a genetic model organism.</a> Physiol Rev. 83, 687-729 (2003).</li><li>Beyenbach, K. W., Skaer, H., Dow, J. 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+developmental,+molecular,+and+transport+biology+of+Malpighian+tubules.">The developmental, molecular, and transport biology of Malpighian tubules.</a> Annu Rev Entomol. 55, 351-374 (2010).</li><li>Donnell, M. J., 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=Hormonally+controlled+chloride+movement+across+Drosophila+tubules+is+via+ion+channels+in+stellate+cells.">Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells.</a> Am J Physiol. 274, 1039-1049 (1998).</li><li>Linton, S. M., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+K+:Cl-+cotransport+and+Na+/K+-ATPase+to+basolateral+ion+transport+in+malpighian+tubules+of+Drosophila+melanogaster.">Contributions of K+:Cl- cotransport and Na+/K+-ATPase to basolateral ion transport in malpighian tubules of Drosophila melanogaster.</a> J Exp Biol. 202, 1561-1570 (1999).</li><li>Rheault, M. R., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+epithelial+K(+)+transport+in+Malpighian+tubules+of+Drosophila+melanogaster:+evidence+for+spatial+and+temporal+heterogeneity.">Analysis of epithelial K(+) transport in Malpighian tubules of Drosophila melanogaster: evidence for spatial and temporal heterogeneity.</a> J Exp Biol. 204, 2289-2299 (2001).</li><li>Donnell, M. J., Dow, J. A., Huesmann, G. R., Tublitz, N. J., Maddrell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separate+control+of+anion+and+cation+transport+in+malpighian+tubules+of+Drosophila+Melanogaster.">Separate control of anion and cation transport in malpighian tubules of Drosophila Melanogaster.</a> J Exp Biol. 199, 1163-1175 (1996).</li><li>Cabrero, P., 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=Chloride+channels+in+stellate+cells+are+essential+for+uniquely+high+secretion+rates+in+neuropeptide-stimulated+Drosophila+diuresis.">Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis.</a> Proc Natl Acad Sci U S A. 111, 14301-14306 (2014).</li><li>Torrie, L. S., 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=Resolution+of+the+insect+ouabain+paradox.">Resolution of the insect ouabain paradox.</a> Proc Natl Acad Sci U S A. 101, 13689-13693 (2004).</li><li>Rodan, A. R., Baum, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+NKCC+Ncc69+is+required+for+normal+renal+tubule+function.">The Drosophila NKCC Ncc69 is required for normal renal tubule function.</a> Am J Physiol Cell Physiol. 303, 883-894 (2012).</li><li>Ianowski, J. P., Christensen, R. J., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na++competes+with+K++in+bumetanide-sensitive+transport+by+Malpighian+tubules+of+Rhodnius+prolixus.">Na+ competes with K+ in bumetanide-sensitive transport by Malpighian tubules of Rhodnius prolixus.</a> J Exp Biol. 207, 3707-3716 (2004).</li><li>Naikkhwah, W., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+stress+alters+fluid+and+ion+transport+by+Malpighian+tubules+of+Drosophila+melanogaster:+evidence+for+phenotypic+plasticity.">Salt stress alters fluid and ion transport by Malpighian tubules of Drosophila melanogaster: evidence for phenotypic plasticity.</a> J Exp Biol. 214, 3443-3454 (2011).</li><li>Donini, A., 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=Secretion+of+water+and+ions+by+malpighian+tubules+of+larval+mosquitoes:+effects+of+diuretic+factors,+second+messengers,+and+salinity.">Secretion of water and ions by malpighian tubules of larval mosquitoes: effects of diuretic factors, second messengers, and salinity.</a> Physiol Biochem Zool. 79, 645-655 (2006).</li><li>Maddrell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+by+Malpighian+Tubules+of+Rhodnius+movements+of+Ions+and+Water.">Secretion by Malpighian Tubules of Rhodnius movements of Ions and Water.</a> J Exp Biol. 51, 71-97 (1969).</li><li>Maddrell, S. H., Overton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+sodium+transport+and+fluid+secretion+by+ouabain+in+an+insect+malpighian+tubule.">Stimulation of sodium transport and fluid secretion by ouabain in an insect malpighian tubule.</a> J Exp Biol. 137, 265-276 (1988).</li><li>Williams, J. C., Beyenbach, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+secretagogues+on+Na+and+K+secretion+in+the+Malpighian+tubules+of+Aedes+Aegypti+(L).">Differential effects of secretagogues on Na and K secretion in the Malpighian tubules of Aedes Aegypti (L).</a> J Comp Physiol. 149, 511-517 (1983).</li><li>Maddrell, S. H., O'Donnell, M. J., Caffrey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+haemolymph+potassium+activity+during+initiation+and+maintenance+of+diuresis+in+fed+Rhodnius+prolixus.">The regulation of haemolymph potassium activity during initiation and maintenance of diuresis in fed Rhodnius prolixus.</a> J Exp Biol. 177, 273-285 (1993).</li><li>Messerli, M. A., Kurtz, I., Smith, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+optimized+Na++and+Cl-+liquid+membranes+for+use+with+extracellular,+self-referencing+microelectrodes.">Characterization of optimized Na+ and Cl- liquid membranes for use with extracellular, self-referencing microelectrodes.</a> Anal Bioanal Chem. 390, 1355-1359 (2008).</li><li>Ianowski, J. P., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+ion+transport+mechanisms+during+fluid+secretion+by+Drosophila+Malpighian+tubules:+Na++recycling,+Na+:K+:2Cl-+cotransport+and+Cl-+conductance.">Basolateral ion transport mechanisms during fluid secretion by Drosophila Malpighian tubules: Na+ recycling, Na+:K+:2Cl- cotransport and Cl- conductance.</a> J Exp Biol. 207, 2599-2609 (2004).</li><li>Neufeld, D. S., Leader, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+characteristics+of+ion+secretion+in+malpighian+tubules+of+the+New+Zealand+alpine+weta+(Hemideina+maori).">Electrochemical characteristics of ion secretion in malpighian tubules of the New Zealand alpine weta (Hemideina maori).</a> J Insect Physiol. 44, 39-48 (1997).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The Theory and Practice of Drosophila Genetics. , (1997).</li><li>Jayakannan, M., Babourina, O., Rengel, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+measurements+of+Na++fluxes+in+plants+using+calixarene-based+microelectrodes.">Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes.</a> J Plant Physiol. 168, 1045-1051 (2011).</li><li>Wu, Y., Schellinger, J. N., Huang, C. L., Rodan, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypotonicity+Stimulates+Potassium+Flux+through+the+WNK-SPAK/OSR1+Kinase+Cascade+and+the+Ncc69+Sodium-Potassium-2-Chloride+Cotransporter+in+the+Drosophila+Renal+Tubule.">Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule.</a> J Biol Chem. 289, 26131-26142 (2014).</li><li>Blumenthal, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+tyramine+signaling+by+osmolality+in+an+insect+secretory+epithelium.">Modulation of tyramine signaling by osmolality in an insect secretory epithelium.</a> Am J Physiol Cell Physiol. 289, 1261-1267 (2005).</li><li>Dow, J. A., Maddrell, S. H., Davies, S. A., Skaer, N. J., Kaiser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+the+nitric+oxide-cGMP+signaling+pathway:+the+control+of+epithelial+function+in+Drosophila.">A novel role for the nitric oxide-cGMP signaling pathway: the control of epithelial function in Drosophila.</a> Am J Physiol. 266, 1716-1719 (1994).</li><li>Dube, K., McDonald, D. G., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transport+by+isolated+anterior+and+posterior+Malpighian+tubules+of+Drosophila+melanogaster:+roles+of+sequestration+and+secretion.">Calcium transport by isolated anterior and posterior Malpighian tubules of Drosophila melanogaster: roles of sequestration and secretion.</a> J Insect Physiol. 46, 1449-1460 (2000).</li><li>Efetova, M., 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=Separate+roles+of+PKA+and+EPAC+in+renal+function+unraveled+by+the+optogenetic+control+of+cAMP+levels+in+vivo.">Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo.</a> J Cell Sci. 126, 778-788 (2013).</li><li>Rheault, M. R., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+cation+transport+by+Malpighian+tubules+of+Drosophila+melanogaster:+application+of+two+novel+electrophysiological+methods.">Organic cation transport by Malpighian tubules of Drosophila melanogaster: application of two novel electrophysiological methods.</a> J Exp Biol. 207, 2173-2184 (2004).</li><li>Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+much+of+a+good+thing:+how+insects+cope+with+excess+ions+or+toxins+in+the+diet.">Too much of a good thing: how insects cope with excess ions or toxins in the diet.</a> J Exp Biol. 212, 363-372 (2009).</li><li>Cheng, C. J., Truong, T., Baum, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney-specific+WNK1+inhibits+sodium+reabsorption+in+the+cortical+thick+ascending+limb.">Kidney-specific WNK1 inhibits sodium reabsorption in the cortical thick ascending limb.</a> Am J Physiol Renal Physiol. 303, 667-673 (2012).</li><li>Cheng, C. J., Yoon, J., Baum, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STE20/SPS1-related+Proline/alanine-rich+Kinase+(SPAK)+is+Critical+for+Sodium+Reabsorption+in+Isolated+Perfused+Thick+Ascending+Limb.">STE20/SPS1-related Proline/alanine-rich Kinase (SPAK) is Critical for Sodium Reabsorption in Isolated Perfused Thick Ascending Limb.</a> Am J Physiol Renal Physiol. , (2014).</li></ol>

The authors have nothing to disclose.

L&#39;uso del saggio Ramsay, insieme con elettrodi ione-specifico, permette la misura dei tassi di secrezione di fluido e flussi ionici in isolato insetti malpighiani (renali) tubuli. Venti o più tubuli possono essere dosati in un momento, consentendo un throughput più elevato rispetto al dosaggio dei singoli tubuli in vitro microperfused. Inoltre, gli elettrodi ione-specifico consentono la determinazione delle concentrazioni di ioni all&#39;interno del fluido secreto in situ, limitando errori che po...

<html> Renale trasporto di ioni epiteliale sottende organismal iono- e osmoregolazione. La Drosophila melanogaster Malpighi (renale) tubulo offre un&#39;opportunità senza precedenti per studiare i meccanismi molecolari del trasporto di ioni epiteliale. Ciò è dovuto alla combinazione delle potenti genetica di Drosophila, accoppiati con l&#39;accessibilità dei suoi tubuli renali allo studio fisiologico. Il saggio Ramsay, dal nome del ricercatore che ha aperto la strada alla tecnica 1, misura i tassi di secrezione di fluidi da isolate tubuli malpighiani, ed è stato fondato nel Drosophila nel 1994 da Dow e colleghi 2. Questo ha spianato la strada a ulteriori studi utilizzando Drosophila strumenti genetici, come il sistema GAL4-UAS 3,4, per definire vie di segnalazione specifici delle cellule che regolano la secrezione di fluidi. Un esempio include segnalazione di calcio in risposta ad un ormone peptidico 5, tra molti altri 6,7. ve_content &quot;&gt; una combinazione di tecniche genetiche e studio fisiologico classica ha dimostrato che la produzione di urina nella mosca avviene attraverso la secrezione di un potassio ricco cloruro fluido dal segmento principale del tubulo. Ciò avviene attraverso la secrezione transepiteliale parallelo di cationi, soprattutto K + ma anche Na +, attraverso la cella principale e Cl -. secrezione attraverso la cella stellate 8-12 La capacità di misurare separatamente transepiteliali K + e Na + flussi consente una caratterizzazione più dettagliata dei meccanismi di trasporto rispetto alla misurazione della secrezione di fluido alone. Ad esempio, in unstimulated tubuli Drosophila, Na + / K + -ATPasi inibitore ouabain non ha alcun effetto sulla secrezione di fluido 2, anche quando il suo assorbimento nelle cellule principali è inibito dal trasportatore anionico organico inibitore taurocolato 13. Tuttavia, Linton e O&#39;Donnell ha mostrato che ouabain depolarizzail potenziale di membrana basolaterale, e aumenta Na + flusso 9. Come mostrato nelle Rappresentante dei risultati, abbiamo replicato questi risultati, e ha mostrato che K + flusso è contemporaneamente diminuito 14; l&#39;aumento del flusso di Na + e diminuita K + flusso avere effetti opposti sulla secrezione di liquidi, con conseguente nessun cambiamento netto della secrezione. Così, ci sono due soluzioni a &quot;paradosso ouabaina&quot;, cioè, l&#39;osservazione iniziale che uabaina non ha alcun effetto sulla secrezione di fluido nel tubulo Drosophila:. Primo, in tubuli stimolato, l&#39;effetto di ouabain sulla secrezione di fluido non è evidente a causa il suo assorbimento da parte del trasportatore anionico organico 13; e in secondo luogo, nei tubuli unstimulated, ouabain ha opposti effetti sulla transepiteliale Na + e K + flusso, con conseguente nessun variazione netta secrezione di fluidi (vedi Rappresentante dei risultati e rif. 9). Pertanto, il ruolo primario della Na + / K + -ATPase in tubuli non stimolati è quello di ridurre la concentrazione intracellulare di Na + per generare un gradiente di concentrazione favorevole per Na + -coupled processi di trasporto attraverso la membrana basolaterale. Infatti, misurando separatamente Na + e K + flussi, abbiamo dimostrato che tubuli manca la cotrasportatore fly sodio-potassio-2-cloruro (NKCC) sono diminuiti K transepiteliale + flusso, senza ulteriore calo dopo ouabaina aggiunta, e nessun cambiamento in transepiteliale Na + Flusso 14. Questi risultati supportati nostra conclusione che Na + di entrare nella cellula attraverso la NKCC viene riciclato attraverso il Na + / K + ATPasi. In un altro esempio, Ianowski et al. Ha osservato che l&#39;abbassamento bagno K + concentrazione da 10 a 6 mm è diminuito transepiteliale K + flusso e aumento transepiteliale Na + flusso nel tubuli da Rhodnius prolixus, senza variazione netta secrezione di fluidi <sup&gt; 15. Diversi effetti sulla Na + K + flusso e flusso attraverso tubuli larvali sono stati osservati anche nei tubuli Drosophila in risposta alle diverse diete sale 16 e in due specie di zanzare in risposta ad allevamento salinità 17. La sfida più grande nella misurazione del flusso transepiteliale di ioni nella preparazione dosaggio Ramsay è la determinazione delle concentrazioni di ioni all&#39;interno del fluido secreto. Questa sfida è stata accolta con soluzioni diverse, tra cui la fiamma photometery 18, l&#39;uso di ioni radioattivi 19, ed elettrone sonda lunghezza d&#39;onda spettroscopia dispersiva 20. Queste tecniche richiedono trasferimento della goccia liquido secreto a uno strumento di misura della concentrazione di ioni. Poiché il volume di fluido secreto dalla non stimolato Drosophila tubulo è piccolo, tipicamente ~ 0,5 Nl / min, questo pone una sfida tecnica e introduce anche errore se parte del fluido è secretoperso al momento del trasferimento. Al contrario, l&#39;uso di elettrodi ione-specifico permette la misura dell&#39;attività ionica (da cui la concentrazione di ioni può essere calcolato) in situ. Il protocollo attuale è stato adattato da quella utilizzata da Maddrell e colleghi per misurare transepiteliale K + flusso attraverso il tubulo Rhodnius utilizzando valinomicina come il K + ionoforo 21, e descrive anche l&#39;uso di un -butylcalix 4- ter [4] acido arene-tetraacetico tetraetile estere basato Na + -specifica elettrodi specifici ioni di litio caratterizzata da Messerli et. al. 22. Elettrodi Ion-specifici sono stati utilizzati anche per misurare le concentrazioni di ioni nel fluido secreto dalle tubuli malpighiani nel test Ramsay negli adulti 9,23 e larvale 16 Drosophila melanogaster, la Nuova Zelanda Alpine Weta (Hemideina maori) 24 e 17 zanzare. Qui, descriviamo in dettaglio l&#39;uso del Ramsaydire di misurare i tassi di fluido secrezione nei tubuli malpighiani da Drosophila melanogaster, nonché l&#39;uso di elettrodi ione-specifico per determinare le concentrazioni di K + e Na + nel liquido secreto e quindi il calcolo dei flussi transepiteliale di ioni. Una panoramica del test è fornito in Figura 1. <img alt="Figura 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Figura 1. Schema del Malpighi tubuli e la Ramsay test con l&#39;uso di ioni a specifici elettrodi per misurare concentrazioni di ioni. Questa figura illustra la configurazione per il saggio Ramsay. (A) Ogni mosca ha quattro tubuli, un paio di tubuli anteriori e una coppia di tubuli posteriori, che galleggiano nella cavità addominale circondato da emolinfa. In ogni coppia, i due si uniscono a tubuli l&#39;uretere, che poi svuota l&#39;urina all&#39;incrocio tra midgut e hindgut. I tubuli sono ciechi a tempo indeterminato. L&#39;urina viene generato attraverso il segmento principale del fluido-secernente (mostrato in rosso), e scorre verso l&#39;uretere e fuori nell&#39;intestino. Dopo la dissezione, la coppia tubulo è dissociata dall&#39;intestino da sezionare l&#39;uretere. (B) La coppia di tubuli viene poi trasferita in una goccia di soluzione salina in un bagno ben del piatto dosaggio. Uno dei due tubuli, qui definito il &quot;ancoraggio tubulo,&quot; è avvolto attorno ad un perno di metallo ed è inerte. L&#39;altro è il tubulo tubulo secernente. Il segmento iniziale (che non secernono liquido) e il segmento principale del tubulo secernono rimangono entro la goccia di soluzione salina balneazione. Ioni e spostare l&#39;acqua dalla salina di balneazione e nel lume del tubulo del segmento principale, e poi si muovono verso l&#39;uretere, come accadrebbe in vivo. Il segmento inferiore (blu) è fuori dalla salina di balneazione e quindi inerte. Poiché l&#39;uretere è tagliato, il liquido secreto emerge come una gocciolina dall&#39;estremità tagliata dell&#39;uretere. Tegli secreto fluido gocciolina allarga nel tempo la secrezione continua, e il suo diametro viene misurato con un micrometro oculare. Uno strato di olio minerale impedisce l&#39;evaporazione del fluido secreto. Gli elettrodi di riferimento specifici e ioni misurano la concentrazione di ioni di liquido secreto. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td><td>May be purchased from multiple distributors</td></tr><tr><td>Petri dish, polystyrene, 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Specific brand is not important</td></tr><tr><td>Petri dish, polystyrene, 35 mm x 10 mm</td><td>Corning Life Sciences</td><td>Fisher 08-757-100A</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Blades #1</td><td>Fine Science Tools</td><td>10011-00</td><td>Specific brand is not important; use appropriate sharps precautions</td></tr><tr><td>Needle, 30 G x 1/2</td><td>Becton Dickinson</td><td>305106</td><td>Use appropriate sharps precautions</td></tr><tr><td>Minutien pins, black anodized, 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Stereomicroscope with ocular micrometer</td><td>Nikon</td><td>SMZ800</td><td>Specific brand is not important; this is given as an example</td></tr><tr><td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td><td>Hobby shop</td><td></td><td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td></tr><tr><td>Glass cutting tools (glass cutter, glass cutting pliers)</td><td>Hobby shop</td><td></td><td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td></tr><tr><td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0042</td><td></td></tr><tr><td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0044</td><td></td></tr><tr><td>Nitric acid, 70%</td><td>Sigma</td><td>438073</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td></tr><tr><td>Cimarec 7 in x 7 in hotplate</td><td>Fisher</td><td>11675911Q</td><td>Specific brand is not important; caution when heated</td></tr><tr><td>Selectophore dichlorodimethylsilane</td><td>Sigma</td><td>40136-1ML</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Two-step vertical pipet puller</td><td>Narishige</td><td>PC-10</td><td>Other pipet pullers can be used; this is given as an example</td></tr><tr><td>Glass petri dish, 150 mm diameter x 15 mm height</td><td>Fisher</td><td>08-748E</td><td>Specific brand is not important; only one dish needed</td></tr><tr><td>World Precision Instruments E210 1 mm micropipette storage jar</td><td>Fisher</td><td>50-821-852</td><td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td></tr><tr><td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td><td>Fisher</td><td>S161-500</td><td>Indicating silica useful for determining whether silica gel retains desiccating ability</td></tr><tr><td>World Precision Instruments MicroFil, 34G</td><td>Fisher</td><td>50-821-914</td><td>May be available from other distributors.</td></tr><tr><td>1 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309659</td><td>May be available from other distributors.</td></tr><tr><td>3 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309657</td><td>May be available from other distributors.</td></tr><tr><td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td><td>Cole-Parmer</td><td>UX-30600-02</td><td>Specific brand is not important</td></tr><tr><td>Female Luer Locking Connector</td><td>4 Medical Solutions</td><td>ADC 9873-10</td><td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td></tr><tr><td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td><td>Fisher</td><td>14-179-110</td><td>Specific brand is not important</td></tr><tr><td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td><td>Fisher</td><td>14171208</td><td>Specific brand is not important</td></tr><tr><td>Modeling clay</td><td></td><td></td><td>Specific brand is not important</td></tr><tr><td>Selectophore potassium ionophore I, cocktail B</td><td>Sigma</td><td>99373</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Selectophore sodium ionophore X</td><td>Sigma</td><td>71747</td><td>Sodium ionophore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td></tr><tr><td>Selectophore 2-nitrophenyl octyl ether</td><td>Sigma</td><td>73732</td><td></td></tr><tr><td>Selectophore sodium tetraphenylborate</td><td>Sigma</td><td>72018</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Schneider&#039;s Drosophila medium</td><td>Life Technologies</td><td>21720024</td><td></td></tr><tr><td>High impedance electrometer</td><td>World Precision Instruments</td><td>FD223a</td><td></td></tr><tr><td>Microelectrode holder 1 mm with 45&amp;deg; body, vented, with handle</td><td>Warner Instruments</td><td>64-1051</td><td></td></tr><tr><td>Microelectrode holder 1 mm with straight body, vented</td><td>Warner Instruments</td><td>64-1007</td><td></td></tr><tr><td>Silver wire</td><td>Warner Instruments</td><td>64-1318</td><td></td></tr><tr><td>Micromanipulators, pair</td><td>Leitz</td><td></td><td>Various brands/models will work; this is an example</td></tr><tr><td>Faraday cage</td><td>Technical Manufacturing Corporation</td><td>81-334-03</td><td>This is an example; any Faraday cage will work</td></tr><tr><td>Single gooseneck fiberoptic light</td><td>Nikon</td><td></td><td>Specific brand is not important</td></tr><tr><td>mineral oil</td><td>Fisher</td><td>BP-2629</td><td>Specific brand is not important</td></tr><tr><td>forceps, Dumont #5 with Biologie tip</td><td>Fine Science Tool</td><td>11295-10</td><td>May be available from other distributors.</td></tr></tbody></table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

Modulazione di trasporto renale di ioni epiteliale permette organismi di mantenere l&#39;omeostasi ionica e osmotico di fronte alle diverse condizioni esterne. La Drosophila melanogaster Malpighi (renale) tubulo offre un&#39;opportunità senza precedenti per studiare i meccanismi molecolari del trasporto di ioni epiteliale, a causa delle potenti genetica di questo organismo e l&#39;accessibilità dei suoi tubuli renali allo studio fisiologico. Qui, descriviamo l&#39;uso del test Ramsay di misurare i tassi di secrezione di fluido dal isolato mosca tubuli renali, con l&#39;uso di elettrodi ione-specifico per misurare le concentrazioni di sodio e potassio nel fluido secreto. Questo test consente di studio fluidi transepiteliale di ioni e flussi di ~ 20 tubuli alla volta, senza la necessità di trasferire il fluido secreto di apparecchiatura separata per misurare concentrazioni di ioni. Geneticamente tubuli distinti possono essere analizzati per valutare il ruolo di specifici geni nei processi di trasporto. Inoltre, il bsalina athing può essere modificato per esaminare gli effetti delle sue caratteristiche chimiche, o farmaci o ormoni aggiunti. In sintesi, questa tecnica permette la caratterizzazione molecolare dei meccanismi di base di trasporto ionico epiteliale nel tubulo Drosophila, nonché regolazione di questi meccanismi di trasporto.

Le figure 7 e 8 dimostrano che l&#39;uso del saggio Ramsay con elettrodi specifici ioni per misurare concentrazioni K + e Na + possono distinguere geneticamente e farmacologicamente distinto K + e flussi Na +, informazioni che non sono catturati misurando i tassi di secrezione di fluido da solo. Figura 7 mostra che diminuiscono la secrezione di fluidi nei tubuli da mosche trasportano un null mutazione omozigotica nel NKCC è guidato da una dim...

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Questo protocollo descrive l&#39;uso del test Ramsay di misurare i tassi di secrezione di fluido dal isolate Malpighian (renale) tubuli da Drosophila melanogaster. Inoltre, l&#39;uso di elettrodi ione-specifico per misurare le concentrazioni di sodio e potassio nel fluido secreto, consentendo il calcolo del flusso transepiteliale di ioni, è descritto.

L&#39;utilizzo del Ramsay Assay misurare la secrezione di fluidi e Ion Flux Prezzi in<em&gt; Drosophila melanogaster</em&gt; Tubulo Malpighi

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

use-ramsay-assay-to-measure-fluid-secretion-ion-flux-rates-drosophila

Research

JoVE Journal

Biology

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

11.8K Views.  University of Texas Southwestern Medical Center.  Questo protocollo descrive l'uso del test Ramsay di misurare i tassi di secrezione di fluido dal isolate Malpighian (renale) tubuli da Drosophila melanogaster. Inoltre, l'uso di elettrodi ione-specifico per misurare le concentrazioni di sodio e potassio nel fluido secreto, consentendo il calcolo del flusso transepiteliale di ioni, è descritto. 

Video: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

La registrazione simultanea dei segnali di calcio da parte dei neuroni identificati e comportamento alimentare di Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Ricerca

Neuroscienze

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Metodi per test Drosophila Comportamento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Analisi per l'integrità della barriera del cervello edelico nella Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Biologia dello sviluppo

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

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

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

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

Misurare risposte fisiologiche di<em&gt; Drosophila</em&gt; Neuroni sensoriali a Lipid feromoni utilizza Live Calcio Imaging

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

Immunologia e infezioni

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

Il capillare alimentatore Assay Misure assunzione di cibo in<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Quantificazione dell'assunzione di macronutrienti in uno schermo neuronale termogenetico usando larve di Drosophila

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Misura della larvali attività nel<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Biologia

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Ottica pH quantificazione di intracellulare in Drosophila melanogaster anatomista Epithelia con pH indicatore fluorescente geneticamente codificato

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Studiare l'attività dei neuropeptidi e di altri regolatori del sistema escretore nella zanzara adulta

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ

Source: Rossano, A. J. and Romero, M. F. <a href="https://www.jove.com/video/55698/" target="_blank">Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator</a>. J. Vis. Exp. (2017).
This article describes the dissection of the anterior Malpighian tubules (MT) from a female Drosophila fly and an example protocol from Rossano et al. (2017) in which the MTs are prepared for live imaging in a perfusion chamber.

L'utilizzo del Ramsay Assay misurare la secrezione di fluidi e Ion Flux Prezzi in

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