Measuring anesthetic resistance in Drosophila by VAAPR

Abstract

Volatile anesthetics are compounds which are commonly used to induce a reversable loss of consciousness (LOC) in animals. The molecular mechanism of how anesthetics induce LOC is largely unknown. However, observations have been made which show that there are genetically-encoded traits which influence the effective concentration of anesthetics in the inducement of LOC. Despite this longterm observation, little progress has been made in identifying genes involved in anesthetic sensitivity. One reason for this is that many techniques to test anesthetic sensitivity are technically challenging and are inhibitory for high-throughput studies. Here we introduce a technique for testing volatiles and aerosols with positional recording (VAAPR), a method which allows for high-throughput testing of the effect of anesthetics and other aerosolized drugs using Drosophila. Using VAAPR we show that the enzyme phospholipase D (PLD) significantly shifts the concentration of diethyl ether, chloroform, and isoflurane needed to induce LOC in Drosophila. We also show that PLD is required for a paradoxical hyperactivity phenotype. We expect that this technique will allow for additional genes to be found which control anesthetic sensitivity as well as other behavioral phenotypes.

Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture

“…If the lipid disruption we observed with HCQ in lung and kidney cells is acting through the same membrane-mediated pathway as shown for general anesthetics, then we expect that HCQ also releases the anesthetic-sensitive protein phospholipase D2 (PLD2) from GM1 clusters. Anesthetics such as xenon, chloroform, isoflurane, propofol, and diethyl ether all displace PLD2 from GM1 clusters to activate an anesthetic pathway 13,61 .…”

Studies on the mechanism of general anesthesia

“…To monitor sedation in vivo, we recorded single-animal measurements (activity and position) of Drosophila melanogaster (fruit fly) in a vertically mounted chamber (Fig. 6A) (40). Flies are a convenient model since they only have one pld gene (41).…”

“…Anesthesia in D. melanogaster. Anesthesia in flies was applied by volatiles and aerosols administered with positional recording (VAAPR) (40) in custombuilt narrow-width vertical chambers placed in front of a camera to monitor the fly's positions with or without the chloroform treatment. Wildtype and PLD null flies were gently loaded into the designated chamber using mouth aspiration and the hoses used for compound delivery were attached to the chamber.…”

Hydroxychloroquine: mechanism of action inhibiting SARS-CoV2 entry

“…To test HCQ for anesthetic-like properties, we treated HEK293T cells with 50 μM HCQ, labeled GM1 lipids and the protein phospholipase D2 (PLD2) with (CTxB, a pentadentate toxin that labels GM1 lipids and anti PLD2 antibody respectively). Phospholipase D2 (PLD2) is an anesthetic sensitive enzyme that translocates out of GM1 rafts in response to general anesthetics xenon, chloroform, isoflurane, propofol, and diethyl ether and provides a live cell test for disruption of lipid rafts 13 38 .…”

“…Phospholipase D2 (PLD2) is an anesthetic sensitive enzyme that translocates out of GM1 rafts in response to general anesthetics xenon, chloroform, isoflurane, propofol, and diethyl ether. Furthermore, mutant flies lacking PLD2 are resistant to anesthesia 10,31 and thus provides a test for anesthetic like effects in the membrane.…”