Year: 2012
Type: article
Abstract: VOLATILE general anesthetics are simple organic compounds that rapidly and reversibly suppress responsiveness to external stimuli in all animals while leaving basal functions intact. These properties have rendered anesthetics indispensable in the clinic and made them attractive tools for studying the control of arousal. One obstacle to progress is that, despite considerable effort over many years, the molecular pathways through which volatile anesthetics act remain largely unknown. In vitro studies have identified many proteins whose activity is affected by clinical concentrations of volatile general anesthetics,1but validating these candidates in vivo has proven difficult.Studies in genetic model organisms such as worms, flies, and mice have been instrumental in identifying novel targets of volatile anesthetics and have provided in vivo validation of candidates identified in vitro .2–6These mutants illustrate several features of the molecular pathways mediating anesthesia. First, voltage-insensitive (“leak”) ion channels, which act to hyperpolarize neurons, have repeatedly appeared in genetic screens for targets of volatile anesthetics,2,3,5,7suggesting that they act, at least in part, through changes in voltage and resistance across the plasma membrane. Second, the response to each anesthetic compound is affected differently by a given mutant, suggesting the presence of agent-specific pathways.2,8Finally, mutations in these genes reduce but do not eliminate sensitivity to volatile anesthetics, indicating that additional anesthetic targets remain unidentified.The ryanodine receptor (Ryr), a large-conductance channel that mediates release of Ca2+from internal stores, is known to be activated by volatile anesthetics, especially halothane.9,10However, the study of the interaction between halothane and Ryr has been largely limited to muscle, where halothane potently induces malignant hyperthermia, a pathologic condition defined by inappropriate activation of Ryr in susceptible humans and swine.11Despite the clear connection between Ryr and halothane, the role of neurally expressed Ryr in the immobilizing effects of halothane is unexplored.In this study, we use Drosophila genetics to test the hypothesis that Ryr mediates halothane anesthesia. We find that dRyr is a major determinant of halothane’s anesthetic effects in flies and that these effects are mediated by expression of dRyr in the nervous system, but not muscle.All flies were reared on cornmeal-molasses medium and maintained at 25°C in a 12-h light–dark cycle. The following fly strains were used in this study: dRyr k04913 , Appl-GAL4 , nrv2-GAL4 , cn 1 l(2)44Fp 1 bw 1 sp 1 /SM6a , cn 1 l(2)44Fl 1 bw 1 sp 1 /SM6a , l(2)44Fa 3 /CyO , l(2)44Ff 1 /CyO , l(2)44Fg 1 /CyO , l(2)44Fh 1 /CyO , and l(2)44Fj 1 /CyO (Drosophila Stock Center, Bloomington, IN); dRyr GS21220 (Drosophila Genetic Resource Center, Kyoto, Japan); and UAS-dRyr RNAi (10844R-3, National Institute of Genetics, Japan). UAS-GCaMP3 was a generous gift from the Howard Hughes Medical Institute (Janelia Farm, Ashburn, VA); elav-GAL4 was a generous gift from Ravi Allada, Ph.D. (Professor, University of Chicago, Chicago, IL); repo-GAL4 was from Chi-Hon Lee, Ph.D. (Senior Investigator, National Institute of Child Health and Human Development, Bethesda, MD); MHC-GAL4 was from Benjamin White, Ph.D. (Senior Investigator, National Institute of Mental Health, Bethesda, MD); and ShakB-GAL4 and Canton-S were from the lab stock collection. dRyr k04913 , dRyr GS21220 , dRyr δ25 , elav-GAL4 , nrv2-GAL4 , and UAS-dRyr RNAi were made congenic with Canton-S by outcrossing for seven generations. As described previously,12dRyr k04913 was homozygous lethal. However, the lethality was not rescued by the genomic duplication dRyr 24D03 , indicating that it resulted from a closely linked mutation outside the dRyr locus.Behavioral assays of anesthesia are subject to a number of confounding factors, including genetic background and subtle environmental variations. To avoid such confounding factors, all comparisons were made within experiments run in parallel over the same time and only between groups matched for genetic background.The deletion mutant dRyr δ25 was generated by excising the region between transposons P{XP}d03686 and P{XP}d03830 (Exelixis Stock Collection, Boston, MA), using flippase-mediated recombination at flippase recombination target sites present in these elements.13The deletion was verified using polymerase chain reaction amplification and DNA sequencing.The genomic duplication dRyr 24D03 was generated by integrating the P[acman] clone CH321-24D03 (Bellen Laboratory, Baylor College of Medicine, Houston, TX) into the VK33 docking site on the third chromosome via φC31-mediated site-specific integration.14UAS-dRyr-V5 was generated by cloning a 15.6-kb PacI-PsiI fragment from DRyR-15B (kindly provided by Daniel Cordova, M.Sc., Senior Biochemist, Dupont Crop Protection, Newark, DE) into the pUAST vector, and injecting the construct into Canton-S flies that carried the w 1118 mutation. All injections were performed by Rainbow Transgenic Flies, Inc. (Camarillo, CA).The genomic DNA libraries from both control and mutant strains used in this study were constructed and sequenced in accordance with the manufacturer’s protocols (Illumina, San Diego, CA). Briefly, 1 μg of genomic DNA was sheared by sonication, end-repaired, A-tailed, adapter-ligated, size-fractionated by gel electrophoresis, and amplified by polymerase chain reaction. Paired-end 101-cycle sequencing on a HiSeq 2000 instrument (Illumina) yielded an average of 20 million reads (range, 13–30 million reads) equivalent to approximately 29-fold coverage (range, 19- to 43-fold). Reads were aligned to the Drosophila melanogaster reference genome (dm3; Berkeley Drosophila Genome Project Release 5) with BFAST.15SAMTools16was used to call sequence variants, and nonsynonymous mutations were identified using ANNOVAR.17To generate a list of unique candidate mutations, we filtered sequence variants in the mutant strains against those present in the control strain. This process revealed that only dRyr contained sequence variants in all of the dRyr alleles carrying the ethylmethanesulfonate mutations. Pairwise alignments were carried out with mammalian Ryr isoforms using BLASTP.After flies were tested in the reactive climbing assay, 200 fly heads or 50–100 whole flies from each genotype were used to prepare homogenates. The samples were homogenized in 100–200 μl of homogenization buffer (0.25 M sucrose, 10 mM Tris, and 1 mM EDTA) and protease inhibitors (Roche, Indianapolis, IN). The homogenates were centrifuged for 10 min at 1,000g at 4°C. The supernatant was centrifuged again for 40 min at 48,000g at 4°C to extract membrane protein. The pellet was resuspended (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, and protease inhibitors), and 20 to 30 μg of protein was processed, electrophoresed, and transferred according to the supplier’s instructions (Invitrogen, Carlsbad, CA). Blots were probed with anti-dRyr (rabbit polyclonal, a generous gift from Daniel Cordova, M.Sc., Senior Biochemist, Dupont Crop Protection) at a 1:1,000 dilution, and anti–Na,K-ATPase (mouse mAbα5; Developmental Studies Hybridoma Bank, Iowa City, IA) at 1.3 ng/ml. Secondary antibodies were peroxidase-linked and used according to the supplier’s instructions. Blots were developed with the enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ). For quantification, four independent homogenates were prepared for each genotype, and two aliquots of each were analyzed, yielding eight blots for each genotype. Intensities of dRyr bands were measured and normalized to those of Na,K-ATPase using ImageJ (National Institutes of Health, Bethesda, MD).The reactive climbing assay, also known as the distribution test, was performed as described previously.18Male flies aged 3–7 days were collected and sorted under carbon dioxide anesthesia and allowed to recover for 1 day. Without further anesthesia, flies were loaded into testing vials (10 per vial), consisting of 50-ml tubes perforated to allow gas exchange. Testing vials were equilibrated in a chamber with a constant flow of a fixed concentration of volatile anesthetics—halothane (Sigma, St. Louis, MO), sevoflurane and enflurane (RxElite, Boise, ID), and isoflurane (Baxter Healthcare, Deerfield, IL)—for 35–45 min. After equilibration, the flies were tapped down quickly to the bottom of the vial several times at 30-s intervals. After the last round of tapping, the flies were allowed to for 1 which the at the bottom of the testing vial was were tested with or four concentrations of by with each test, and equilibrated for 35–45 min each flies of the same genotype were tested at the four or concentrations to the of the from down to The process was with In this yielded a of for each of and was performed as described a of 30 flies of each genotype, aged 3–7 were loaded into the tapped to the and given 10 to into a at The flies in each were into the by the This was four times at and at the the flies in each were , is by the following of flies in the of flies in all The was performed to times for each with a generous gift from Daniel Cordova, were from were maintained at in medium and at by approximately prepare for they were in mM NaCl, mM mM 1 mM mM mM 10 mM and mM centrifuged and resuspended in of 1 of the of a in were in for and in for a of 30 min approximately were in tubes and with anesthetics and of the was measured on a NJ). and were out by and by was at the of the by of and as described by for and for of was carried out using concentrations in were to using in internal in the of using the by ShakB-GAL4 The nervous system was from the and were and down on a which the bottom of the chamber in a that into the of a Japan). This allowed for from and the was using a from The was at 1 with the at and the to mM NaCl, mM mM mM 10 mM mM mM sucrose, mM at using a with via were into for min and from of anesthetic in with were via of samples of collected from the using an gas were using of in be at of four to eight per The of each in the of a given was defined as a region of and the average time was the background in outside of was background was was by the at a given time by the average of samples 1 to the to and by to generate a were performed largely as described external was mM NaCl, mM mM mM mM mM sucrose, mM mM The internal by the of and mM mM NaCl, mM 10 mM 1 mM mM mM mM nervous system, in under the control of ShakB-GAL4 , was on a of The region of the in was with and with a were under the of and membrane voltage was using an City, in and were and were using were as described under in and at climbing were largely as described the were into Chicago, and with the down using is the anesthetic the of the and is between for each and the anesthetic concentration at the of the vial of flies was used for four to anesthetic that the of eight concentrations a of The for each of for which the in using the following and were in San CA). For of for were normalized by of with and those with concentrations of anesthetic as and the were with a for were and as for but using that with to halothane concentrations mM were from the by test for the for between two groups were using the a were performed using The of and anesthetics described under dRyr on was performed with The test was used for of the of the were to 1 in a number of Drosophila genome a Ryr at of the role of dRyr in the response to the volatile anesthetic halothane, we used dRyr k04913 , a mutant from the of a of the of dRyr dRyr GS21220 , of dRyr k04913 dRyr GS21220 is homozygous as described previously,12dRyr k04913 is of a linked mutation outside the dRyr under and for the of the transposons in the two we them to the response of the mutants to halothane using a reactive climbing that the following in the presence of these Canton-S flies a in in an in the to with flies at the bottom of the vial at the anesthetic For dRyr GS21220 and dRyr k04913 and the dRyr k04913 GS21220 , the to the indicating resistance to halothane to the The concentrations at which of flies were down were and for dRyr GS21220 dRyr k04913 and dRyr k04913 GS21220 , all of which were that of the control for and In the dRyr GS21220 was also for matched in halothane sensitivity was by in dRyr protein was a of genotype on protein and the that halothane sensitivity the dRyr k04913 GS21220 , dRyr protein dRyr protein in the were between the and dRyr k04913 GS21220 , but to from We that the alleles reduce dRyr but that anesthetic sensitivity between alleles do identified target genes for anesthetic that have a for halothane over anesthetic the presence of agent-specific the of anesthetic of we the of dRyr mutants to and and them to the response to halothane using a was a of anesthetic on the of in with halothane the In dRyr k04913 GS21220 mutants were to and isoflurane with dRyr k04913 in the for and isoflurane that were not from and were the of this on dRyr GS21220 resistance to which was not from that for halothane, but not the response to sevoflurane or dRyr mutants were to the anesthetic effects of halothane to those of volatile dRyr as a of the that halothane the between dRyr dRyr protein and halothane sensitivity we generated flies with the dRyr or The deletion dRyr δ25 was by excising the region between two using recombination recombination The all but the of dRyr and all of , a of This resistance to halothane, both in a and in with the Ryr GS21220 and and In dRyr δ25 which of dRyr , halothane in dRyr δ25 GS21220 mutants halothane which is the but not from dRyr δ25 As be for a dRyr GS21220 the and dRyr δ25 or dRyr δ25 GS21220 dRyr we an additional genomic of dRyr into chromosome 3 to generate the dRyr duplication dRyr 24D03 dRyr 24D03 rescued the of dRyr GS21220 with the in dRyr GS21220 dRyr 24D03 from dRyr GS21220 and and not from the dRyr 24D03 into a in of dRyr , to halothane , a in of dRyr 24D03 suggesting that of the be the response to halothane not from or in that all performed or animals tested for and responsiveness to in the of anesthetic was the for the dRyr mutants was a of dRyr genotype on dRyr protein In dRyr protein was and in dRyr δ25 and dRyr δ25 dRyr GS21220 The in dRyr protein in dRyr 24D03 not that halothane sensitivity dRyr number and protein We that dRyr is a for anesthesia and a target of number in humans is mutations in Ryr genes are that to is in sequence to the mammalian and protein to Ryr indicating that Ryr activity and be across identified a of mutants in region we that of these mutants 3 , l(2)44Fg 1 , l(2)44Fh 1 , l(2)44Fj 1 , and l(2)44Fp 1 the sequence of dRyr of these dRyr alleles contained mutations at to in l(2)44Fa 3 , and at in l(2)44Fj 1 In both mutations a the region the ion and As for were to halothane , dRyr , to matched control l(2)44Ff 1 and and and both to the of dRyr δ25 The of halothane resistance in the mutants appeared to be that of the deletion dRyr δ25 but in genetic of the animals in the two experiments comparisons alleles were mutations, to of in l(2)44Fg 1 , in l(2)44Fh 1 , and in l(2)44Fp 1 All of the mutations were in with dRyr δ25 , two of the mutations are in a region with in under Ryr and for and all to halothane sensitivity was enhanced in dRyr in and and in dRyr and dRyr and and the mutations in dRyr effects on halothane anesthesia in Drosophila , the by an of dRyr the malignant hyperthermia, mutations in are with a condition in which in response to volatile anesthetics, the reactive climbing is on and be by dRyr in muscle, we the site of of dRyr by the expression of RNAi using UAS-dRyr RNAi with the MHC-GAL4 to halothane sensitivity to carrying MHC-GAL4 and flies UAS-dRyr RNAi under the control of the elav-GAL4 and Appl-GAL4 were to halothane and in and suggesting that dRyr is in for halothane UAS-dRyr RNAi expression in with repo-GAL4 also resistance to halothane in indicating that dRyr is in and for halothane anesthesia. by nrv2-GAL4 , which but not in and UAS-dRyr RNAi a but not in further the site of of we to halothane sensitivity in dRyr mutants by a UAS-dRyr in expression of the construct using the and expression in and using nrv2-GAL4 sensitivity to halothane in dRyr k04913 GS21220 mutants in This also dRyr protein expression in the of rescued flies, as by blots of and experiments the that dRyr functions in the nervous system and not to control anesthetic responsiveness of Drosophila In the lethality from UAS-dRyr in with the that homozygous dRyr 24D03 and dRyr δ25 are that or in dRyr expression are of role in described in the that dRyr in the nervous system is for to halothane, but the of halothane to the Drosophila Ryr has not been We in with dRyr the and flow Without halothane, the of in to and with halothane concentrations 1 mM to a in all with of with to the that the response to halothane on internal on at concentrations indicating that the response to halothane to halothane in an basal or of given halothane concentration and were as the of response to in this the for which the response is on dRyr was to the anesthetics, for halothane with of and 1.3 mM for halothane, and to these to and indicating that halothane is sevoflurane or We that molecular between halothane and the dRyr protein the sensitivity of dRyr mutants to halothane anesthesia in Drosophila the effects of halothane on in of Drosophila , we the expression of the using ShakB-GAL4 to halothane with in in an response to halothane that the of halothane the chamber The of this response a with an halothane of which was in response to concentrations of halothane of an additional consisting of a with at or the time of halothane response of to halothane was by mutations in dRyr In for the deletion mutant, dRyr δ25 , and the dRyr , the to mM halothane were by and with a these not 1 and and However, for mutant dRyr , which in the reactive climbing to halothane with indicating that this the response of dRyr to The of dRyr for both reactive climbing and that it is a for halothane response of to provided additional into the of the mutations. to were to those of an with mM the in in dRyr was in the and the response of for dRyr was also to an to that of dRyr the the response to halothane but the response to indicating that the mutation the response of dRyr on the of the This also the lethality of dRyr δ25 , in that the dRyr for a of halothane a was of or that through in the plasma membrane. the membrane on the halothane and both approximately was at the the by with is by a of the suggesting the activation of an molecular and of volatile anesthetic have been the subject of study for This not only from the of anesthesia but also from the that the molecular and pathways that anesthetic effects into the of and has been generated by the that general anesthesia to these the for protein targets of volatile anesthetics has The present study that in Drosophila the ryanodine receptor is to such an anesthetic We that neurally expressed dRyr mediates the response to the volatile anesthetic halothane, with sensitivity to halothane anesthesia on of dRyr mutations in the dRyr that generate and proteins resistance to halothane, mutations that flies to the that the dRyr halothane sensitivity to and that in nervous from dRyr mutants in response to halothane, further the that dRyr is a target of the that the of halothane is to dRyr of as described for dRyr , dRyr GS21220 , dRyr δ25 , or resistance to mutants that are to and dRyr channels, also an additional genomic of dRyr to and experiments that dRyr in and but not muscle, is for to halothane anesthesia and that dRyr expression in these is to anesthetic The anesthetic are from those in malignant hyperthermia, a condition with mutations that halothane of the , in humans and This is by the that of dRyr expression in to the halothane sensitivity of reactive that both the Drosophila Ryr and the mammalian of this that halothane sensitivity is a general of in and neurons, which and halothane induces from is with that dRyr mediates in Drosophila and a that the immobilizing effects of of Ryr is two have been defined for activation and interaction with halothane Ryr by it do by from the or by release from that of internal not in at of halothane, they have been with it has been that halothane in membrane against activation of dRyr by it that halothane Ryr by with of many this that which do not the with We the hypothesis that halothane with the dRyr protein. The of the of in in the of the response of to do not a protein such as which has been to of dRyr by halothane a of in which a of a of the to the response of to halothane with The of the that in the presence of halothane, or that rapidly from the The of on of halothane is with activation of for and of by activation of dRyr to to the anesthetic in and that is a of halothane with of The of Ryr activation is to be an carried by a are but it is to that the and of Ca2+from the a novel to by the plasma membrane which internal external in in channels, such as which are to internal also an additional for the of carbon which is membrane through the of and as an of these and remain to be but the described that the genetic and tools in Drosophila be in between dRyr activity and halothane anesthesia has the between volatile anesthetics and and pathologic with of is also in this that two of the mutations in dRyr that we to sensitivity to halothane are in a region with is to a mutation in , that is with of a condition by dRyr is a to that mutations the of halothane in reactive climbing assay, and the release of suggesting of isoforms and across is the that the in sensitivity to halothane but activation by indicating that the mutant protein is not mutations in neurally expressed Ryr in humans to the effects of In of this halothane activity of malignant in susceptible on of the of anesthesia in genetic model organisms is that, despite the of each volatile anesthetic is to act through of molecular The of targets mediating halothane anesthesia it is the in such as flies and worms, to mammalian anesthesia. In by volatile anesthetics is to be mediated by and such as are of the mammalian and However, the of between the of and is not and the of of molecular targets of anesthetics to be the of the to and the sites of and experiments dRyr expression in of and and it be to dRyr expression in of these to anesthesia on the activity of a or a halothane in that with we have the between and membrane the , of dRyr These for National Institute of Mental Health, National Institutes of Health, Bethesda, and the for of Daniel Cordova, (Senior Biochemist, Dupont Crop Protection, Newark, for the anti-dRyr and (Senior Investigator, National Institute of Mental Health, National Institutes of Health, Bethesda, for the use of National Institute of Mental Health, National Institutes of Health, Bethesda, for and on the Ph.D. (Senior and Ph.D. National Institute of Mental Health, National Institutes of Health, Bethesda, for National Institute of Mental Health, National Institutes of Health, Bethesda, for Benjamin White, Ph.D. (Senior Investigator, National Institute of Mental Health, National Institutes of Health, Bethesda, for use of and in the and (Professor, and Ph.D. National Institute of and National Institutes of Health, Bethesda, for
Source: Anesthesiology
Institutions: Universities at Shady Grove, University of Maryland, College Park, National Institute of Mental Health, National Institute of Mental Health, National Institute of Mental Health, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke
Language: English
Subfield: Anesthesiology and Pain Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2002914386