! ! ! We#thank#referee##3#for#his#favourable#comments#on#our#manuscript.## ! Below#are#detailed#answers#to#his#cri;cisms:# ! i)
!
The& authors& failed& to& define& a& quan3ta3ve& measure& for& the& change& in& the& HOMO& observed& upon& its& interac3on&with&the&specific&molecules.&Hence,&when&the&claim&"biggest&perturba3on"&it&is¬&clear&how& they&reach&this&conclusion.&&
We#agree.#To#make#the#comparisons#between#different#anesthe;cs#somewhat#more#quan;ta;ve,#we#have# now#measured#the#volume#added#to#the#HOMO#by#the#presence#of#the#anesthe;c.#To#compensate#for#the# fact#that#larger#anesthe;c#molecules#have#larger#HOMOs#and#therefore#allow#more#spread,#we#normalise# this#added#volume#by#the#molecular#volume#of#the#anesthe;c.#This#ra;o,#which#we#call#R,#appears#to#give#a# good# heuris;c# for# anesthe;c# ac;on,# and# correctly# predicts# the# high# anesthe;c# potencies# of# Xe,# barbital,# etomidate,#alfaxalone#and#cyclopropane.#It#also#correctly#predicts#that#the#fluorinated#compounds#fluothyl# and#DMPFC#should#be#nonOanesthe;c.#SF6,# however,#though#it#clearly#extends#the#HOMO,#does#not#fit#this# quan;ta;ve#rela;onship.#We#have#edited#sec;ons#3.2.1#and#3.2.2,#and#the#corresponding#figures#10#and#11# to#describe#and#discuss#this#new#data.##Sec;on#4.2.1#in#the#discussion#has#also#been#edited.##
!
ii)& If& the& effect& observed& is& related& to& "electron& current"& it& may& be& affected& also& by& the& proper3es& of& the& LUMO.&&
!
In#the#shortOhelix#model#we#studied,#the#LUMO#is#at#the#opposite#end#of#the#helix,#and#is#unaffected#by#the# presence#of#an#anesthe;c.##
!
3.&The&discussion&on&the&origin&of&the&spin&signal&is¬&transparent.&It&seems&that&if&a&Boltzman&distribu3on& of&spins&among&the&spin&states&is&assumed,&the&signal&cannot&be&explained.&&
!
Our#wording#was#ambiguous.#We#meant#to#say#that#one#possibility#is#that#our#signal#is#due#to#a#reversible# increase# in# the# total# amount# of# spin# [spin# up+spin# down],# in# the# standard# measurement# condi;ons# of# equilibrium#ESR,#i.e.#with#Boltzmann#distributed#spins.#We#discuss#three#possible#places#where#these#added# spins#may#reside:#free#radicals;#metal#ions;#and#as#charge#carriers#in#melanin#and#proteins.##
!
The&authors&men3on&"spin&polariza3on&by&the&magne3c&field"&but&this&is&a&very&general&statement&and&it&is& not&obvious&how&this&effect&results&in&the&signal&observed.&
! We#agree#that#the#wording#was#confusing.#It#has#been#changed.# !
&Recently&spin&polariza3on&was&observed&in&electron&transfer&through&chiral&molecules&(JPCL&3,&2178&(2012)).& Can&this&effect&be&relevant&to&the&studied&system?&&
!
We#are#grateful#to#the#referee#for#poin;ng#out#the#spin#polarisa;on#experiments#due#to#electron#transfer# through#chiral#molecules.#We#became#aware#of#this#work#a_er#the#ini;al#submission#and#are#glad#to#be#able# to# include# it# in# the# discussion# now.# We# think# it# is# a# strong# contender# as# a# possible# mechanism# and# are# designing#experiments#to#test#this#possibility.##
!
NOTE:# It# seems# to# us# that# figure# 12# is# now# made# redundant# by# these# changes,# and# we# have# therefore# omi`ed#it.##
! Minor&comments&& !
1. In&the&introduc3on,&it&is¬&clear&why&the&authors&men3oned&the&olfactory&system.&Is&the&idea&to&present& another& case& where& there& is& no& clear& understanding& what& is& going& on?& I& suggest& that& the& authors& will& omit&this¶graph.&&
!
We# agree# that# this# paragraph# was# confusing# in# this# context.# It# has# been# removed,# and# parts# of# the# last# paragraph#of#the#intro#have#been#moved#to#the#discussion,#the#rest#rewri`en.#
! 2.&Is&there&data&on&the&temperature&dependent&of&the&effect&(at&temperatures&where&the&flies&are&frozen)?&& !
We#did#not#try#this#because#we#were#concerned#that#penetra;on#of#anesthe;c#into#frozen#;ssue#would#be# limited,#and#also#because#the#boiling#point#of#SF6# ,#O64C,#limits#the#temperature#range#before#condensa;on# occurs.#
Electron$spin$and$anesthesia$in$Drosophila
Significance)statement 160$years $a*er$its $discovery,$the$molecular$mechanism$of$general$anesthesia$remains$an$outstanding$ mystery.$A$very$wide$range$of$agents$ranging$from$the$element$Xenon$to$steroids$can$act$as $general$ anesthe=cs$on$all$animals$from$protozoa$to$man,$sugges=ng$a$basic$cellular$mechanism$is$involved.$ In$ this$ paper$ we$ show$ that$ vola=le$ general$ anesthe=cs$ cause$ large$ changes$ in$ electron$ spin$ in$ Drosophila$fruit$flies,$and$ that$the$spin$responses$are$different$in$anesthesiaEresistant$mutants.$We$ propose$ that$ anesthe=cs$ perturb$ electron$ currents$ in$ cells,$ and$ describe$ electronic$ structure$ calcula=ons$on$anesthe=cEprotein$interac=ons$that$are$consistent$with$this$mechanism$and$account$ for$hitherto$unexplained$features$of$generalEanesthe=c$pharmacology.$
1
Electron$spin$and$anesthesia$in$Drosophila
Electron)spin)changes)during)general)anesthesia)in)Drosophila 1 Luca)Turin1§,)E:himios)M)C)Skoulakis1)and)Andrew)P)Horsfield2 1Biochemical$Sciences$research$Centre$“Alexander$Fleming”,$16672$Vari,$Greece 2Department$of$Materials,$Imperial$College$London,$London$SW7$2AZ$UK §to$whom$correspondence$should$be$addressed,
[email protected].$Author’s$present$address:$Ins=tute$of$
Theore=cal$Physics,$Universität$Ulm,$Albert$Einstein$Allee,$89069$Ulm,$Germany.
0D)Abstract We$ show$ that$ the$ vola=le$ general$ anesthe=cs$ Xe,$ SF6,$ N2O$ and$ CHCl3$ cause$ rapid$ increases$ of$ different$magnitude$and$=me$course$in$the$electron$spin$content$of$Drosophila.$With$the$excep=on$ of$ CHCl3$ these$changes$ are$reversible.$ Anesthe=c$ resistant$ mutant$ strains$of$ Drosophila$exhibit$ a$ different$paeern$of$spin$responses$to$anesthe=c.$In$two$such$mutants$the$spin$response$to$CHCl3$ is$ absent.$We$propose$that$these$spin$changes$are$caused$by$perturba=on$of$the$electronic$structure$ of$ proteins$ by$ general$ anesthe=cs.$ Using$ Density$ Func=onal$ Theory,$ we$ show$ that$ general$ anesthe=cs$perturb$and$extend$the$HOMO$ of$a$5Eresidue$alpha$helix.$The$calculated$perturba=ons$ are$ qualita=vely$ in$ accord$ with$ the$ MeyerEOverton$ rela=onship$ and$ some$ of$ its$ excep=ons.$ We$ conclude$that$there$may$be$a $connec=on$between$spin,$electron$currents$in$cells $and$the$func=oning$ of$the$central$nervous$system.$
1D)IntroducGon General$anesthesia $(GA)$ is$ both$ indispensable$and$ fascina=ng.$ Millions$of$ surgical$procedures$are$ performed$each$year,$most$of$which$would$be$unthinkable$if$ general$anesthe=cs$(GAs)$did$not$exist.$ Yet$while$the$first$clinical$anesthesia$with$diethyl$ether$was$reported$over$160$years$ago{Perouansky$ 2012},$the$mechanism$by$which$the$same$GAs$act$on$animals $as$far$apart$in$evolu=on$as$paramecia$ and$man{Oliver$1991}—and$even$plants{Zimmerman$1936}{Okazaki$1993}{DeDeLuccia$2012}—is$s=ll$ unclear.$ In$ 2005,$ general$ anesthesia$ was$ included$ in$ a$ Science$ Magazine$ list$ of$ major$ unsolved$ problems,$in$the$august$company$of$cancer,$quantum$gravity$and$highETc$superconduc=vity${Kennedy$ 2005}.$ Today,$ GA$ remains$ an$ intellectual$ challenge,$ and$ arguably$ one$ of$ the$ few$ experimental$ inroads$to$consciousness{Alkire$2008}{Kulli$1991}{Hameroff$2006}. The$ mystery$ of$ GA$ resides$ in$ a$ uniquely$ baffling$ structureEac=vity$ rela=onship:$ the$ range$ of$ compounds$ capable$ of$ ac=ng$ as$ GAs$ makes $ no$ pharmacological$ sense.$ Adrien$ Albert$ called$ it$ “biological$ac=vity$unrelated$to$structure”{Albert$1985}.$In$number$of$atoms,$the$simplest$of$the$GAs$ is $Xenon{Lawrence$1946}{Cullen$1951},$a$monoatomic$noble$gas,$the$most$complex$ is$alfaxalone$(3E hydroxypregnane$ E11,20Edione),$ a$ 56Eatom$ steroid{Laubach$ 1955},$ spanning$ a$ 35Efold$ range$ in$ molecular$volume.$In$between$lies$a $host$of$molecules$of$ widely$different$structures:$nitrous $oxide,$ halogenated$ compounds$ (SF6,$ chloroform,$ halothane,$ etc),$ strained$ alkanes $ (cyclopropane),$ 1$Author$contribu=ons:$LT$and$EMCS$designed$the$fly$experiments,$LT$and$APH$designed$the$Density$Func=onal$Theory$
calcula=ons.$LT$performed$experiments$and$calcula=ons.$LT,$EMCS$and$APH$analyzed$data$and$wrote$the$ar=cle.$
2
Electron$spin$and$anesthesia$in$Drosophila
phenols{James$ 1980}(propofol),$ ethers$ (diethyl$ ether,$ sevoflurane),$ amides$ (urethane),$ sulfones$ (tetronal),$ pyrimidines $(barbiturates),$etc.$If$ one$adds$gases,$like$dioxygen$and$ nitrogen,$that$ cause$ narcosis$ under$ pressure,$ and$ vola=le$ solvents$ used$ as$ inhala=onal$ recrea=onal$ drugs,$ the$ list$ is$ longer$ s=ll.$ What$ property$ can$ all$these$ molecules$ possibly$ have$in$ common$ that$ causes$ general$ anesthesia$? A$ par=al$ answer$ has$ been$ known$ for$ nearly$ a$ century.* GAs$ are$ lipidEsoluble$ and$ their$ potency,$ regardless$of$structure,$is$approximately$propor=onal$to$lipid$solubility$[with$some$excep=ons{Koblin$ 1994}],$ a$ rela=onship$ known$ as$ the$ MeyerEOverton$ rule{Meyer$ 1899}{Overton$ 1901b}reviewed$ in{Perouansky$2012}.$This$implies,$surprisingly$in$light$of$their$diverse$structures,$that$once$arrived$at$ their$des=na=on,$all$GAs$are$equally$effec=ve.$Accordingly,$since$GAs$dissolve$in$the$oily$core$of$the$ lipid$ bilayer,$ they$ were$ long$ thought$ to$ perturb$ the$ featureless$dielectric$ in$ which$ ion$ channels,$ receptors$ and$ pumps$ are$ embedded{Cantor$ 1997}{Mullins$ 1954}{Seeman$ 1972}{Miller$ 1973}{Lee$ 1976}{Trudell$1977}{Gruner$1991},$though$an$ac=on$on$proteins$could$never$be$ruled$out{Burn$1946} {Featherstone$ 1961}.$ Other$ theories$ were$ also$ proposed,$ involving$ the$ forma=on$ of$ gas$ hydrates{Pauling$ 1964},$ proton$ leaks{Bangham$ 1980},$ hydrogen$ bonds{Chiou$ 1990}{Brockerhoff$ 1990}and$membrane$dipoles{Qin$1995}.$In$the$80s,$however,$following$the$discovery$of$an$effect$of$ anesthe=cs$on$firefly$luciferase{Ueda$1965}Franks$and$Lieb{Franks$1978}first$demonstrated$enzyme$ inhibi=on$by$GAs{Moss$1991},$ then$differences$in$potency$between$GAs$enan=omers{Franks$1991} {Hall$1994}.$This$pointed$to$a$protein$site$of$ac=on,$likely$a$weakly$chiral$hydrophobic$pocket{Franks$ 1997}{Franks$1998}{Franks$1998}{Trudell$1998}.$Indeed,$general$anesthe=cs $are$now$believed$to$act$ on$ proteins{Eckenhoff$ 1997}{Eckenhoff$ 2008}{Sonner$ 2013}and$ have$ now$ been$ seen$ in$ just$ such$ sites$in$protein$structures,$where$they$exert$small$but$definite$effects$on$protein{Bhaeacharya$2000} and$ ion$ channel{Sauguet$ 2013}conforma=on.$ The$MeyerEOverton$ rule$then$ becomes$all$the$more$ surprising,$since$protein$binding$sites$are$usually$highly$selec=ve$for$ligand$shape$and$size. But$if$indeed$both$the$MeyerEOverton$rule$and$ the$FranksELieb$protein$ hypothesis $are$taken$ to$ be$ correct,$a$single$mechanism$should$be$shared$by$all$GAs$at$the$protein$binding$site(s).$Then$the$small$ GAs,$and$ Xenon$ especially,$ dras=cally$ constrain$ the$range$of$ possibility.$ Xenon$ is$uniquely$ slippery$ and$falls$outside$the$normal$confines$of$molecular$recogni=on.$It$has$no$"shape”,$since$it$is$a$perfect$ sphere$of$electron$density.$It$has$no$chemistry$either,$at$any$rate$under$condi=ons$found$in$the$brain.$ However,$and$this$is$the$hitherto$overlooked$star=ng$point$of$the$new$ideas$developed$in$this$paper,$ Xenon$ has$physics:$ like$ many$ other$ elements$ and$ molecules,$ it$ is$capable$of$ facilita=ng$ electron$ transfer$between$conductors:$recall$the$iconic$photograph$of$the$IBM$ logo$wrieen$in$Xe$atoms $in$a$ scanning$ tunnelling$ microscope$ (STM){Eigler$ 1990}.$ Each$ Xe$atom$ is$ a$bump$ because$ it$facilitates$ tunnelling$from$substrate$to$=p,$and$the$=p$must$rise$above$it$to$keep$current$constant{Eigler$1991}.$ Indeed,$ among$ the$many$ molecules$ that$ have$ been$ imaged$ in$ the$STM$ are$ several$GAs$or$ close$ congeners$ other$ than$ Xenon:$ nitrous $ oxide{Watanabe$ 2005},$ phenols{Sakamaki$ 1989},$ ethers{Nishino$2001},$benzene{Yau$1996},$amides{Takeuchi$1996},$pyrimidines{Kim$1996}. Suppose$ then$that$there$exists,$in$one$or$more$proteins$essen=al$to$central$nervous$system$(CNS)$ func=on,$a$hydrophobic$site$lined$with$an$electron$donor$on$one$side$and$an$acceptor$on$the$other.$ When$GAs$enter$the$site,$they$could$connect$donor$to$acceptor$by$crea=ng$ a$pathway$for$electron$ transfer$where$there$was$none.$Tunnelling$conductance$is$such$a$general$property$of$molecules $that$ if$ a$ connec=on$ were$ found$ between$ it$ and$ anesthesia,$ the$ MeyerEOverton$ rule$ would$ follow$
3
Electron$spin$and$anesthesia$in$Drosophila
naturally.$How$would$one$detect$these$currents$in$a$whole$organism?$If$the$electrons$are$unpaired,$ electron$spin$resonance$provides$a$specific,$though$not$par=cularly$sensi=ve$detec=on$method,$the$ only$one$presently$applicable$to$whole$animals.$It$therefore$seemed$interes=ng$to$ask$whether$one$ could$detect$changes$in$electron$spin$during$anesthesia.$
2D)Methods Drosophila:)Drosophila*were$cultured$in$standard$cornmeal$sugar$food$supplemented$with$soy$flour$ and$CaCl2$at$20°–22°C. ESR) measurements:$ An$ Adani$ EMS$ 8400$ [www.adani.by]$ fieed$ with$ a$ temperature$ control$ aeachment$ was$used$ to$ measure$ spin$in$ live$flies.$The$flies$ were$paralyzed$ briefly$ with$ CO2$ and$ approximately$30$flies$dropped$into$a$teflon$tube$3.5$mm$ID$[see$figure$1$le*]$.$The$tube$was$placed$ in$the$RF$cavity.$RF$parameters$were:$frequency$≈$9.4$GHz,$90$mW,$6dB$aeenua=on.$measurements$ at$aeenua=ons$between$3$and$21$dB$showed$an$approximately$linear$rela=onship$to$the$square$root$ of$incident$RF$power$and$no$evidence$of$ satura=on.$The$magne=c$field$was$modulated$at$1000$µT.$ Fixed$magne=c$field$recordings$were$sampled$ at$34.13$samples/second$ for$120$ seconds.$The$ =me$ constant$of$ the$ESR$detector$circuit$is $approximately$10ms.$Magne=c$field$scans$were$done$at$24$s/ mT.$Except$where$otherwise$specified,$the$flies $were$kept$at$6C$ to$immobilize$them.$The$flies$were$ bathed$in$a$stream$of$temperatureEcontrolled$nitrogen$flowing$from$below$with$an$pressure$into$the$ tube$of$ 6$mbar.$The$experiments$typically$ lasted$less$that$30$minutes$a*er$which$control$flies$not$ exposed$to$anesthe=cs$invariably$revived$rapidly.$An$approximate{Eaton$2010}calibra=on$of$ the$ESR$ was$performed$by$weng$ a$small$piece$of$ filter$ paper$with$20µl$of$ an$solu=on$of$1µM$ TEMPOL$in$ water.$The$amplitude$of$the$calibra=on$signal$was$≈$ 3000$arbitrary$units$at$the$gain$sengs$used$in$ the$ con=nuous$ measurements$ reported$ in$ this$ paper.$ The$ data$ was$ processed$ with$ LabView$ [www.ni.com]$and$ploeed$with$Igor$[www.wavemetrics.com]. Density) FuncGonal) Theory) (DFT)) calculaGons:) The$Amsterdam$ Density$ Func=onal$ [www.scm.com]$ Density$Func=onal$Theory$package$was$used$in$the$calcula=ons.$All$structures$were$op=mized$using$ PBE$ func=onal$and$ TZP$basis$set$[helix$ alone]$ or$a$dispersionEcorrected$ PBEED3$func=onal$and$ TZP$ basis$set$[helix+$anesthe=c].$Orbitals$were$then$recalculated$at$the$final$geometry$using$B3LYPETZP.$ Some$semi$ empirical$ preEop=misa=ons$ as$ well$ as$ orbital$ volume$calcula=ons$were$ performed$ in$ MacSpartan$14$[www.wavefun.com].$Calcula=ons$were$done$on$a$12Ecore$Apple$MacPro.$
3D)Results 3.1)Electron)spin)resonance)measurements)in)Drosophila Organic$free$radicals$are$so$reac=ve$that$their$concentra=on$in$living$ cells$is$generally$ found$ to$ be$ subEnanomolar,$ and$ therefore$ unmeasurable$ by$ ESR$ whose$detec=on$ limit$ is$approximately$ three$ orders$ of$ magnitude$higher${Swartz$ 1972a}.$In$ the$ absence$of$ spin$ traps$which$ integrate$the$free$ radical$signal$over$=me,$the$cellular$component$contribu=ng$most$of$the$“free$electron”$spin$signal$ [i.e.$with$a$gEfactor$near$2.0]$will$be$melanins,$which$typically$contain$1018$ spins/g{Blois$1964}.$Flies$ are$heavily$pigmented,$and$we$can$expect$a $melanin$signal$propor=onal$to$pigmenta=on.$This$is$the$ case,$ as$ shown$ in{Trapp$ 1983}and$ replicated$ with$ our$ equipment$ in$ figure$ 2$ [right]$ inset.$ Three$
4
Electron$spin$and$anesthesia$in$Drosophila
strains$of$Drosophila,$namely$normallyEpigmented$wild$type,$and$yellow1white,$and$neuralized,*both$ of$ which$are$devoid$of$ eumelanin$but$s=ll$contain$pheoE$ and$neuroE$ melanins,$exhibit$spin$signals$ propor=onal$to$pigmenta=on.$The$melanin$signal$is$unremarkable,$centered$around$2.0$and$with$a$ width$of$≈10$gauss$(fig$1$inset).$No$spin$traps$were$used$in$these$experiments.$
3.1.1)Spin)increases)reversibly)during)exposure)to)the)general)anestheGc)SF6). Against$this$large$but$on$a$scale$of$minutes$presumably$steady$melanin$background,$we$seek$a$signal$ that$varies$with$anesthesia.$ As$a$first$guess,$we$expect$ it$ to$ also$lie$around$g=2.0,$but$to$likely$ be$ much$smaller$than$the$melanin$signal.$We$therefore$made$highEsensi=vity$measurements $at$a$fixed$ value$of$magne=c$field$corresponding$to$the$posi=ve$peak$of$the$background$spin$signal,$i.e.$≈3340$ gauss$(the$exact$value$varies$slightly$from$experiment$to$experiment$and$is$set$each$=me). Figure$1$shows $a$typical$experiment$illustra=ng$ spin$changes $during$ exposure$to$SF6,$ a$chemically$ inert$anesthe=c$gas$which$was$found$to$give$the$largest$signal$[see$sec=on$3.1.6$for$effects$of$other$ anesthe=cs].$The$dura=on$of$a$single$trace$[4096$points]$is$120$seconds.$Here$approximately$30$flies$ are$ measured$ con=nuously$ in$ a $ fixed$ magne=c$ field$ of$ 3346$ G.$ The$ flies$ are$ ini=ally$ bathed$ in$ nitrogen$at$6$degrees$C.$The$low$temperature$is$necessary$to$make$sure$the$flies$do$not$move,$which$ would$make$the$measurements$impossible.$At$t=24$seconds$SF6$ gas$is$injected$into$the$chamber.$The$ spin$signal$increases$rapidly$ and$stabilizes$at$a$new$ value$approximately$7000$units$higher.$At$t=60$ seconds$N2$ is$readmieed$ to$ the$chamber$ and$ the$ signal$ returns$to$ baseline$with$ a$slower$ =meE course$than$ that$ of$ the$onset.$The$raw$data$(trace$2)$ is$ baselineEcorrected$ by$fing$ a$ line$ to$ the$ period$ immediately$preceding$ exposure$to$ the$anesthe=c$ and$subtrac=ng$ the$line$from$ the$signal$ (trace$ 3);$ the$ signal$ is$ then$ lowEpass$ filtered$ by$ boxEaveraging$ [50$ points](trace$ 4).$ An$ empty$ chamber$ (trace$ 1)$ gives$no$ signal$ and$ slightly$ more* noise$ than$when$flies$ are$present.$ The$noise$ therefore$appears$to$be$largely$instrumental$in$origin$and$lies $at$high$frequencies$compared$to$those$ of$the$signal.
Figure) 1:$ Le::$ Experimental$ setup$ for$ con=nuous$ spin$ measurements$ on$ live$ flies.$ Approximately$ 28E32$ Drosophila$are$housed$ in$a$PTFE$tube$ inserted$ into$ a$quartz$tube$and$posi=oned$ in$the$RF$resonator$ cavity$of$ an$ESR$spectrometer.$A$hollow$PE$plug$at$the$boeom$allows$the$temperatureEcontrolled$cooling$gas$coming$up$ from$below$ [blue$ arrow]$ to$enter$ the$fly$tube$ if$the$ other$ end$is$ le*$open.$Anesthe=cs$ are$ introduced$in$ the$ chamber$ by$connec=ng$a$syringe$to$the$top$plug$and$cannula.$The$total$chamber$volume$is$about$1.5$ml.$Right:$ Con=nuous$measurements$of$spin$at$a$fixed$value$of$magne=c$field$corresponding$to$the$peak$of$the$intrinsic$ spin$resonance$[inset].$In$each$2Eminute$trace$the$sample$chamber$is$filled$with$SF6$for$36$seconds$from$the$24$
5
Electron$spin$and$anesthesia$in$Drosophila
second$=me$mark.$At$ other$ =mes$temperature$controlled$ nitrogen$at$ 6C$flows$through$the$ chamber.$Trace$1:$ empty$chamber,$signal$unaffected$by$SF6.$Traces$2E4,$recording$from$W1118$wildEtype$flies.$The$raw$data$[trace$ 2]$ is$baseline$ corrected$ by$fing$a$straight$line$ to$ the$ period$ before$ SF6$ exposure$ and$subtrac=ng$that$ line$ from$ the$ en=re$ trace,$ yielding$ trace$ 3.$ Trace$ 3$ is$ then$ smoothed$ with$ a$ 50Epoint$ box$ filter$ [trace$ length$ is$ 4096points]$to$ remove$ instrumental$ highEfrequency$noise.$Note$ that$ the$ noise$ level$ is$ highest$ in$ the$empty$ chamber.$
3.1.2)The)anestheGcDinduced)spin)change)is)a)resonance. To$check$that$the$anesthe=cEinduced$spin$change$is$not$due$to$a$change$in$baseline$due$either$to$a$ broad$background$resonance$or$to$a$mistuning$of$ the$RF$cavity,$we$exposed$the$flies $successively$to$ SF6$ at$ values$of$ the$ magne=c$ field$ close$ to$ the$ peak,$ the$ crossover$ point$ and$ the$ trough$ of$ the$ background$signal.$A$ resonance$centered$around$2.0$should$be$posi=ve$at$the$peak,$nearEzero$near$ crossover$and$nega=ve$in$the$trough.$Figure$2$le*$ shows$that$ his$is$the$case.$ Exposures$to$SF6$ at$ 3346,$ 3349$and$ 3356$gauss$give$signals$that$are$ posi=ve,$close$to$ zero$ and$ nega=ve$ respec=vely.$ Accordingly,$ subtrac=ng$ the$background$ spin$signal$ (inset$ A,$ black$ trace)$ from$the$spin$during$ SF6$ exposure$ (inset$ A,$ red$ trace)$ yields$ a$ clearly$ resonant$ signal$ whose$ amplitude$ matches$ well$ the$ traces$taken$at$the$different$magne=c$fields.$Responses$to$other$anesthe=cs$[see$below]$ exhibit$the$ same$ sign$reversal$when$measured$ in$ the$ trough$[not$shown].$ We$conclude$ that$ the$ spin$ change$ caused$ by$ SF6$ exposure$ is$ a$ genuine$ spin$ resonance.$ A$ typical$ SF6$ signal$ of$ 7000$ units$ therefore$ represents$an$increase$in$spins$in$the$cavity$of$the$order$of$2.8$!013$spins.
Figure) 2:$Le::) Inset) A:$Spin$ signal$ from$ ≈30$W1118$ wild$ type$ flies$ before$ (black$trace)$ and$ a*er$ (red$ trace)$ exposure$to$SF6.$B:$Difference$trace$resul=ng$from$the$two$traces$in$ panel$A,$i.e.$the$spin$ signal$introduced$ by$ SF6.$C:$four$traces$measured$at$fixed$values$of$magne=c$field$corresponding$to$points$1$to$4$on$trace$B,$i.e.near$ the$top$[1],$close$to$ the$crossover$ [2],$halfway$down$ [3],$and$just$past$the$boeom$ peak$[4].$The$amplitude$of$ the$traces$ in$ C$ matches$ the$ amplitudes$ expected$ from$ B,$showing$ that$ the$ spin$ changes$ seen$ in$ fixedEfield$ con=nuous$measurements$do$indeed$reflect$ an$ underlying$resonance.$The$other$anesthe=cs$behave$similarly.$ Right:)Effect$ of$melanin$content$in$different$fly$strains$on$anesthe=cEinduced$ spin$changes.$Inset:$intrinsic$ spin$ signals$of$W1118$wildEtype,$and$two$ “pale”$fly$strains,$YellowEWhite$[red$trace]$and$Neuralized$[blue$trace],$in$ which$ eumelanin$ is$ absent.$ The$ intrinsic$ spin$ signal$ of$ the$ pale$ strains$ is$ much$ smaller,$as$ expected.$Main$ panel:$Percentage$ change$ in$spin$in$ response$to$ SF6$ in$ the$three$strains.$If$the$signal$were$due$ to$melanin,$it$ should$ either$ be$ reduced$ in$amplitude$or$ disappear$ altogether.$Instead,$it$ increases$in$ inverse$propor=on$ to$ the$intrinsic$spin$level,$showing$that$the$two$are$independent.
6
Electron$spin$and$anesthesia$in$Drosophila
3.1.3)The)SF6Dinduced)spin)change)is)independent)of)eumelanin)content There$are$many$“yellow”$Drosophila$strains$in$which$eumelanin$is$absent.$We$used$two,$yellow1white$ and$neuralized.*As$expected$from$previous$work{Trapp$1983},$the$background$spin$signal$in$yellow1 white*and*neuralized*is$reduced$to$about$one$third$of$the$wild$type$spin$signal$(figure$3$right,$inset).$ The$change$in$spin$during$exposure$to$SF6,$however,$does$not$scale$with$the$eumelanin$content$but$ instead$becomes$propor=onately$larger$as$total$spin$ is$reduced$ in$the$yellow$ mutants,$ sugges=ng$ that$it$ is$not$due$to$eumelanin.$We$cannot$rule$out$that$the$spin$signal$reflects$an$increase$in$the$ spin$of$pheoE$or$neuromelanin,$because$we$have$to$date$been$unable$to$obtain$strains$of$completely$ white$flies.$
3.1.4)ProperGes)of)the)SF6Dinduced)spin)signal The$measurements$above$were$made$with$a$large$modula=on$depth$of$10$gauss,$to$maximise$signal$ amplitude.$This$is$achieved$at$ the$expense$of$ lineshape$resolu=on.$ In$ order$to$measure$lineshape$ op=mally,$we$first$determined$the$rela=onship$between$microwave$power$and$amplitude$of$the$SF6E induced$signal.$The$background$melanin$signal$was $previously$shown$not$to$be$near$satura=on$[see$ methods].$Here$we$check$for$the$powerEintensity$rela=onship$of$the$addi=onal$anesthe=c$inducedE spin.$ The$ results$ are$ shown$ in$ figure$ 3A.$ Repeated$ exposures$ to$ SF6$ at$ different$ power$ sengs$ ranging$from$E6dB$[22.5$mW$RF$incident$power]$to$E18dB$[1.4$mW$RF]$showed$a$monotonic$rela=on$ between$signal$amplitude$and$RF$power.$The$results$of$seven$such$experiments$done$both$in$wildE type$and$YellowEWhite$strains$are$shown$in$figure$3B.$They$agree$reasonably$well$with$the$predicted$ powerEsignal$rela=onship$ far$ from$ satura=on,$ namely$ a$ signal$propor=onal$ to$ the$ square$root$ of$ incident$power.$Measurements$made$at$6bB$were$highly$repeatable$over$a$scale$of$ tens$of$minutes,$ whereas $at$higher$power$values$[3dB$and$above]$showed$some$instability$and$increase$in$noise.$It$is$ not$clear$ whether$ this$is$due$to$the$flies$warming$ up$ enough$ to$ move$or$ whether$ the$hea=ng$ is$ sufficient$to$cause$damage.$
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signal$amplitude$5.103
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RF$a