Dampened neural activity and abolition of epileptic-like activity in cortical slices by active ...

12 Pages·2014·2.14 MB·English

Dampened neural activity and abolition
OPEN
of epileptic-like activity in cortical slices
SUBJECTAREAS:
by active ingredients of spices
NATURALPRODUCTS
EXCITABILITY
MaurizioPezzoli1,AbdeladimElhamdani1*,SusanaCamacho1,2,JulieMeystre1,
Received StephanieMichligGonza´lez2,JohannesleCoutre2,3&HenryMarkram1
17June2014
Accepted 1LaboratoryofNeuralMicrocircuitry.BrainandMindInstitute.E´colePolytechniqueFe´de´raledeLausanne.Switzerland,2Laboratory
19September2014 ofPerceptionPhysiology.Nestle´ResearchCenter.Lausanne,Switzerland,3OrganizationforInterdisciplinaryResearchProjects,The
UniversityofTokyo,Tokyo,Japan.
Published
31October2014
Activeingredientsofspices(AIS)modulateneuralresponseintheperipheralnervoussystem,mainly
throughinteractionwithTRPchannel/receptors.ThepresentstudyexploreshowdifferentAISmodulate
neuralresponseinlayer5pyramidalneuronsofS1neocortex.TheAIStestedareagonistsofTRPV1/3,
Correspondenceand
TRPM8orTRPA1.Ourresultsdemonstratethatcapsaicin,eugenol,menthol,icilinandcinnamaldehyde,
requestsformaterials butnotAITCdampenthegenerationofAPsinavoltage-andtime-dependentmanner.Thiseffectwas
shouldbeaddressedto furthertestedfortheTRPM8ligandsinthepresenceofaTRPM8blocker(BCTC)andonTRPM8KOmice.
M.P.(maurizio. Theobservableeffectwasstillpresent.Finally,theinfluenceoftheselectedAISwastestedoninvitro
gabazine-inducedseizures.Resultscoincidewiththeaboveobservations:exceptforcinnamaldehyde,the
[email protected])
sameAISwereabletoreducethenumber,durationoftheAPburstsandincreasetheconcentrationof
gabazineneededtoelicitthem.Inconclusion,ourdatasuggeststhatsomeoftheseAIScanmodulate
*CurrentAddress: glutamatergicneuronsinthebrainthroughaTRP-independentpathway,regardlessofwhethertheneurons
arestimulatedintracellularlyorbyhyperactivemicrocircuitry.
A1Science4kids,
5603Dowgate,CT
RockvilleMD20851, Spices are dried seeds, fruit, roots, bark, or other vegetable substances that are added to food in small
US. quantitiestoprovideflavororcolor,oraspreservativesthatkillorpreventthegrowthofharmfulbacteria1.
Examplesrelevanttothisstudyincludechilli,cloves,menthol,mustard,wasabi,radishandcinnamon.In
manycultures,theseandotherspicesalsoplayanimportantroleintraditionalmedicine2,wheretheyareusedas
analgesicsandlocalanaesthetics,aswellastoemulatethermosensation3,4.
SomeActiveIngredientsofSpices(AIS)bindtoTransientReceptorPotential(TRP)ionchannels-afamilyof
mainlynon-selectivesodium/calciumchannelswithgreaterdiversityinactivationmechanismsandselectivity
thananyotherfamilyofionchannels.TRPchannelshavebeenshowntobeinvolvedwithsignaltransductionin
vision,taste,olfaction,hearing,touch,andthermo-andosmo-sensation5,6,7,8.AsubsetofTRPchannelsisreported
torespondtomoleculespresentinspices.Forexample,TRPV1isactivatedbyheat($43uC)butalsorespondsto
capsaicin4,theactivecompoundofchilipepper; eugenol9;anandamide10(anendocannabinoid);camphor11,a
topical analgesic; and piperine, the pungent ingredient in black pepper12. All these compounds produce a
psychophysiological sensation of heat or burning. TRPM8 is activated by moderately cool temperatures
(,23–28uC)13,14andbycompoundssuchasmenthol15,eucalyptol16,andicilin17,whichproduceacoolingsen-
sation.TRPA1,whichissometimesco-expressedwithTRPV118,19,respondstoavastrangeofagonistspresentin
spices including allicin and diallyl disulfide (garlic), cinnamaldehyde (cinnamon), gingerol (ginger), a-SOH
(Sichuanpeppers)andcanabinoids20.
MoststudiesofspicesandtheirinteractionwithTRPchannelshavefocusedonsensoryneuronsandonthe
sensoryterminalsoftargetorgans7.However,ithasalsobeenobservedthattheAIScancrossthebloodbrain
barrier,modulatingtheactivityofthecentralnervoussystem(CNS).Inparticular,capsaicin21,linalool22,and
menthol23arereportedtoinhibitseizurebehaviourininvivomodelsofepilepsy.
Here,wehavebuiltonthiswork,systematicallyinvestigatingtheeffectofAISontheelectricalbehaviourof
singleneuronsandneuronalnetworks.Wefindthatfouractiveingredientsofspices(capsaicin,eugenol,menthol,
cinnamaldehyde)andonesyntheticanalog(icilin)exertavoltageandtime-dependentinhibitoryeffectonsingle
cellactivity,andalmostallofthesamemoleculesinhibitburstingactivityincorticalpreparations.Weshowthat
theseeffects,unliketheeffectsofspicesonsensoryneurons,areindependentofTRPchannels.Spicesandtheir
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homologuesaresuccessfulininhibitingneuronalfiringandprevent- firing thresholds were significantly higher than in the control
ingthereleaseofglutamateinavoltageandtime-dependentmanner. conditionandlateAPamplitudeswerelower(Fig2D2).Thedrop
Our data suggests that spices and their homologues could play a inamplitudeofAPswaslargestwhenthecellwashighlydepolarized.
usefulroleinthetreatmentofexcitotoxicityandassociatedclinical Thisimpliesthattheeffectofmentholisvoltage-dependent. With
conditionssuchasstroke,Alzheimer’sdisease,Huntington’sdisease, this type of stimulation ligands of TRPV1/3 had a stronger effect,
andbrainandspinalcordinjuries,tomentionafew24.Weobserve leadinginsome casesto atotalabolitioninthegenerationofAPs
thatspices,ascommonfoods,haveafavorablesafetyprofile,andwe (datanotshown).Nosignificantchangeswereobservedintheinput
proposefurtherworktoexploretheirtranslationalpotential. resistance (Fig 3A–C) or in the waveforms of the initial APs at
rheobase(Fig3D–E).
Results Taken together, these data show that menthol exerts a general
dampeningeffectonneuralactivity.Theyalsoshowthatthiseffect
Dampeningoffiringactivityinsingleneurons.Wefoundthatfive
is voltage and time-dependent. The data collected for capsaicin,
out of the six AIS (capsaicin, eugenol, menthol, icilin,
cinnamaldehyde) induced large, qualitatively similar, voltage and eugenol,mentholandicilinwerequalitativelysimilar,althoughexact
time-dependent changes intheelectrical activityofsingle neurons parametervaluesvariedfromcompoundtocompound(Table1).By
(Fig 1). These AIS all induced drastic changes in the number and contrast,AITCproducednosignificanteffect,andcinnamaldehyde
amplitude of APs generated at a given voltage. The effect was affectedmorefeatures(i.e.:inputresistanceandAPwidth)(Table1).
observable in all neurons that received the treatment. As an Acontrolstudyusingslicesfromlateralamygdalashowednomajor
example of the detailed effects, we can examine changes in the differencescomparedtocorticalslices(datanotshown).Anegative
electrophysiological behaviour of recorded cells after perfusion controlusingaCSF1DMSOshowednosignificantdifferencewith
withmenthol250 mM(n59)(Fig2).Uponstimulationwithaset respecttoaCSFonly(datanotshown).
ofconsecutivelyincreasingsquarecurrentpulses,themeandropin
theamplitudeofAPsbetweenthestartandtheendofeachpulsewas Role of TRP channels. All of the AIS studied act as potent TRP
significantlylargerafterperfusion(Fig2A,2B).Thetotalnumberof channel agonists and one possible explanation for the effects
APsgenerated afteragivennumber oftrialswasalsosignificantly described above is that AIS act directly on their corresponding
lower after perfusion, with the largest gap in later trials when the TRP receptors expressed in the brain. To test this hypothesis, we
membranevoltagewashighest.ThelastAPrecordedforeachpulse investigated this possibility for menthol and icilin known to be
wasalsobroaderandthetimebetweenthebeginningofthepulseand potentagonistsofTRPM8.TRPM8ligandshavebeendescribedas
thelastAPwasshorter(Fig2C).Stimulationwithsteadilyincreasing havingtheleastcrossreactionwithotherTRPchannels(notthecase
current, leading to increased depolarization (Fig 2D), showed that for TRPV and TRPA1 ligands25,26), which allows for more precise
Figure1|Voltagerecordingsofsinglelayer5pyramidalneuronsoftheprimarysomatosensorycortex.Comparedvoltagetracesofdifferentsinglecell
recordingsathighdepolarization,incontrolsituation(black)andinthepresenceofaparticularAIS(red).Tracesaregroupeddependingofthe
correspondingAISreceptorsaffinity:-TRPV1/3ligands:1.-Capsaicin25mM;2.-Eugenol200mM.-TRPM8ligands:1.-Menthol250mM;2.-Icilin
50mM.-TRPA1ligands:1.-AITC500mM;2.-Cinnamaldehyde300mM.ExceptforAITCallAIStestedtendtoaffectthegenerationtheAPsathigh
depolarizationlevels.
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Figure2|RecordedtracesofstimulielicitingAPaffectedbyextracellularmenthol250mMandcorrespondinganalysis.Singlecellvoltagerecordingsin
controlconditions(black)andinthepresenceofmenthol250mM(red)fromS1somatosensorycortexlayer5pyramidalneurons.Thefeaturesextracted
wereaffectedbythepresenceoftheAIS.Voltagerecordings(A)underpulsestimuli,asetof11successivetrialsofincreasingtwo-secondlongsquare
currentsteps.Tracesshowactionpotentialfiringinthecontrolsituation(top,black)andundertheeffectsofmenthol250mM(bottom,red).Theaction
potentialamplitudedroprate(B)wasobtainedfromeachcorrespondingcurrentstepforbothconditions(1)theamplitudedropratesforeach
conditionwereaveragedandplotted(2)(n59average6S.E.;p,0.005assessedbyStudent’st-test).Furtheranalysiswasperformedonthelastaction
potentialofeachofthe11trialspercondition(C1).Thesefeatureswereplottedagainsttheircorrespondingtrials,forbothconditions(C2)(average6
S.E.;n59).Rampstimuli(D1)wasperformedprovidingameasurefortheactionpotentialthreshold(D2,Top)(p,0.005;assessedbyStudent’st-test)
andtheactionpotentialamplitudedroprate(D2,Bottom)(p,0.05;assessedbyStudent’st-test)plottedforbothconditions(average6S.E.;n59).
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Figure3|Analysisofmenthol250mMeffectsonneuralactivitythroughcurrentstimulation.TherecordingsofI/Vanalysissetofstimuli(A)provided
voltagevalues(toptraces)atpeakandatsteadystateforcontrol(black)andinthepresenceofmenthol250mM(red;circleforpeakvoltagevaluesand
squareforsteadystate.)ateachlevelofcurrentinjected(bottomtraces).Datawasplottedforeachcellandinputresistancewasobtainedfrombothvoltage
levelsforbothconditions(B)bylinearfit.Whencompared,(C)theinputresistanceatpeakvoltageandatsteadystateshownostatisticaldifference
betweencontrol(black)andinthepresenceofmenthol250mM(red)(n59average6S.E.;assessedbyStudent’st-test).Actionpotentialwaveform
analysisfeatures,amplitudeandwidth,wereobtainedfromhighsampling(20kHz)voltagetraces(Dtoptraces)fromcontrol(black)andmenthol
250mM(red)conditionswhileapplyingsquarecurrentpulses(bottomtraces).Valueswereplottedandcompared(E)presentingnostatisticaldifference
(n59average6S.E.;assessedbyStudent’st-test).
Table1|AnalyzedfeaturesoftheeffectofdifferentTRPligandsintheelectrophysologicalpropertiesinS1L5pyramidalneurons
APDRp(mV/pA) Lp#AP(nmbr) APDRr(mV/pA) Thres(mV) APAmp(mV) APWidth(ms) IRpeak(MV) IRs.s.(MV) N
Ctrl 20.4160.08 37.262.2 228.262.9 241.861.4 92.362.6 1.2260.08 97.068.2 74.766.6
Mntl 22.0260.32 13.664.3 269.467.5 236.961.4 89.362.5 1.2260.08 96.069.2 74.067.2
t-test 0.0009 0.001 0.0001 0.00003 0.004 0.485 0.349 0.368 9
Ctrl 20.3460.17 40.064.5 220.162.6 237.161.4 91.661.7 0.9560.03 74.968.3 56.267.5
Icln 21.1860.37 23.967.1 232.266.0 232.962.0 87.962.0 1.0260.05 65.169.3 52.369.3
t-test 0.009 0.042 0.013 0.004 0.010 0.085 0.015 0.137 7
Ctrl 20.0260.01 41.861.6 3.462.4 243.261.9 73.864.7 1.2560.05 84.467.3 70.266.4
Cpcn 20.7860.23 9.864.4 229.5610.0 230.363.3 72.562.1 1.3760.14 87.469.1 72.467.6
t-test 0.099 0.0003 0.058 0.076 0.387 0.211 0.335 0.363 5
Ctrl 20.3860.07 54.062.6 223.863.5 241.860.6 81.061.7 0.8760.03 56.563.2 44.263.18
Egnl 23.7961.21 4.061.4 252.6617.4 230.861.2 78.861.9 1.0460.09 49.863.1 41.363.3
t-test 0.106 0.000001 0.079 0.005 0.180 0.052 0.0134 0.141 7
Ctrl 20.2860.07 51.363.2 217.464.2 242.360.7 73.162.6 1.0160.03 64.662.5 51.162.1
Cnld 20.5360.09 26.963.5 226.663.3 238.960.8 73.162.4 1.1860.04 76.564.0 61.763.2
t-test 0.0003 0.0005 0.023 0.00014 0.499 0.003 0.021 0.011 8
Ctrl 20.2760.13 41.862.3 220.864.8 239.662.2 75.364.2 0.9660.05 74.9610.3 61.069.0
AITC 20.3360.31 28.364.7 225.168.1 241.862.6 75.964.5 1.1160.09 95.0616.3 84.4615.4
t-test 0.393 0.003 0.168 0.008 0.315 0.015 0.026 0.017 6
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controloftheTRPpathwayfortheseagonists.TRPV1receptorshave blocker30. We found that in the absence of AIS (the first phase of
been identified in the central nervous system27,28,29 and TRPA1 as ourexperimentalprocedure),dosesofgabazineof1 mMandhigher
well20, but no TRPM8 has been localized in the mouse brain. The reliablyinducedrapidburstingbehaviour(Fig7Aand7C).However,
general TRP blocker, Ruthenium Red and the specific TRPV1 perfusionofcorticalsliceswithcapsaicin,eugenol,mentholoricilin,
blocker capsaizepine on their own affected the firing behaviour of diminishedorabolishedburstingwithlowerdosesofgabazineand
theglutamatergicneurons,renderingthemmorereactive(datanot drasticallyreducedburstingathigherones.Theeffectformenthol
shown). This made them unsuitable for a clear evaluation of a 250 mM when the bursting protocol was being performed was
possible participation of TRP channels when exposed to AIS. observedinsingle cellrecordings (Fig7Band7D).Perfusion with
BCTC aspecific TRPM8 blocker,showed little ornoeffect onthe menthol completely abolished bursting for doses of gabazine
parameter measured by our electrophysiological protocol (Fig 4, ,10.0 mM. Washout partially restored the original bursting
Table2)whencomparedtocontrolsituation. behaviour underlining that the abolition of bursting is due to the
ThefirstsetofexperimentswasperformedwithBCTCinS1L5 actionofmenthol.Repetitionoftheprotocolwithcapsaicin,eugenol
pyramidal neurons (n 5 16). We were able to observe the same andicilin,inducedqualitativelysimilareffects.Cinnamaldehydeand
qualitativeeffectsasinthepreviousstudy(Table2).Mentholshowed AITC on the other hand produced an inverse effect leading to an
qualitatively the same effect in the absence and in the presence of intensificationofburstingactivity(Fig8,Table4and5).
BCTC15 mM(Fig4).Mentholhadnomeasurableeffectonthewave
form of APs or on input resistance (Fig 4C and D respectively). Discussion
However,eveninthepresenceoftheblocker,perfusionwithmenthol Wehaveshownthatperfusionofbrainsliceswithcapsaicin,eugenol,
wasassociatedwithvariationsinthelastAPwhentheneuronwas menthol, icilin dampens the activity of single neurons, especially
stimulatedwithsuccessivelyincreasingstepsofsquarecurrentpulses whenneuronsaremaintained inadepolarizedcondition.Further,
(Fig4A1).Acorrelationwasalsoobservedbetweenthepresenceof wehaveshownthatperfusionwiththeseAIScompletelyorpartially
theAISand withafasterdroprateofAPamplitudewhenmeasured abolishes bursting behaviour in an in vitro model of seizure. This
in square pulses stimulations orincreasing ramp (Fig 4A2 and 4B resultiscompatiblewithpreviousreportsthatseveralAIS(capsai-
right, respectively) as well as withhigher firing thresholds(Fig 4B cin21, linalool22, menthol23), inhibit epileptic-like behaviour in vivo
left). Results for icilin were qualitatively similar (Table 2). These andinvitro31.
resultsimplythatthedampeningeffectsofmentholarenotdue,or Interestingly, several different AIS produce qualitatively similar
atleastnotexclusivelydue,totheireffectonTRPM8.Similarresults effects both on single neurons and on larger-scale network beha-
wereobservedinslicesfromlateralamygdala. viour.Thissuggeststheexistenceofacommonmechanismofaction
GiventhatsomeoftheobservedeffectsoftheAISwereweakerin thatcouldbesharedbyotherAISandsomeoftheirsyntheticanalo-
the presence of the blocker (Table 3), we repeated the study with gues.Onthebasisoftheparametersanalyzed,theeffectselicitedon
TRPM8ligandsusingTRPM8KOmice(n514).Asacontrol,the glutamatergicneuronsappeartobemoresimilarbetweenTRPV1/3
recordingswererepeatedonWTlittermates(n515)(Table3).Aset andTRPM8ligandsthanthoseproducedbyTRPA1one.
ofvoltagetracesinresponsetothedifferenttypesofstimuliofasingle Theeffectobservedcouldbedescribedasadropintheprobability
recording of a TRPM8 KO mouse are shown in the control, while ofgeneratingsubsequentAPs.Iftheneurons,inthepresenceofthe
applying menthol 250mM and in washout conditions (Fig 5). The AIS, were stimulated by a square pulse from holding membrane
effectoftheAISontheAPsathighdepolarizationlevelsisevident.A potential(270 mV)tosuprathresholdvalues,theynormallyelicited
moredetailedanalysisofthefeaturesobtainedwithmenthol250mM atleastoneAP.TheprobabilityofobservingsubsequentAPswhile
perfusion for TRPM8 KO and WT litter mates was plotted (Fig 6). thedepolarizationlasteddependedonthevoltage(themoredepo-
TherewaslittleornodifferencebetweenKOandWT.TheAISeffect larized,thelowertheprobability)andhowlongitwasmaintained
remainedmostlythesame.Similaractivitywasobservedforicilinat (the longer the neuron was depolarized, the probability also low-
50mM(Table 3).Weconcludethattheobservedeffectsofmenthol ered).Thiscontrastedwiththefactthat oncetheneuron’s voltage
andicilinareindependentoftheiractiononTRPM8. was brought back to 270 mV the condition was somehow reset.
Thus, when the neuron was again depolarized, it was able to fire
Abolitionofburstingbehaviourinaninvitromodelofepilepsy. again.(Fig2).Whenstimulatedwithaconstantandgradualincrease
ThedampeningeffectoftheselectedAISonneuronfiringprovidesa ofcurrent,thresholdlevelbecamemoredepolarizedinthepresence
possibleexplanationforpreviousobservationsthatsomeAISinhibit oftheAIS.Insomecases(TRPV1/3ligands)noAPwasobserved.
epileptic-likeburstingbehaviourinslices.Toinvestigatethiseffect These behaviours could indicate that AIS affect the inactivation
further we used an in vitro model of epilepsy using a GABA phaseinthefastsodiumcurrents.
A
Table2|AnalyzedfeaturesoftheeffectofdifferentTRPM8ligandsintheelectrophysologicalpropertiesinS1L5pyramidalneuronsinthe
presenceandabsenceofaTRPM8receptorblocker(BCTC15 mM)
APDRp(mV/pA) Lp#AP(nmbr) APDRr(mV/pA) Thres(mV) APAmp(mV) APWidth(ms) IRpeak(MV) IRs.s.(MV) N
Ctrl 20.4160.08 37.262.2 228.262.9 241.861.4 92.362.6 1.2260.08 97.068.2 74.766.6
Mntl 22.0260.32 13.664.33 269.467.5 236.961.4 89.362.5 1.2260.08 96.069.1 74.067.2
t-test 0.0009 0.0002 0.0001 0.00003 0.004 0.485 0.349 0.368 9
BCTC 20.3360.12 44.067.1 228.264.6 240.061.3 93.662.2 0.9960.06 90.9610.4 73.569.8
BCTC1Mntl 21.0860.29 24.568.9 245.269.2 236.261.4 88.962.5 0.9860.05 91.269.4 72.869.0
t-test 0.021 0.042 0.009 0.0001 0.039 0.397 0.488 0.445 8
Ctrl 20.3460.17 40.064.5 220.162.6 237.161.4 91.661.7 0.9560.03 74.968.3 56.267.5
Icln 21.1860.37 23.967.1 232.266.0 232.962.0 87.962.0 1.0260.05 65.169.3 52.369.3
t-test 0.009 0.042 0.013 0.004 0.010 0.085 0.015 0.137 7
BCTC 20.2760.05 48.364.1 218.664.3 240.861.2 93.962.5 0.9860.07 68.167.5 52.366.7
BCTC1Icln 20.6660.21 29.865.8 220.365.5 238.261.1 93.463.4 1.0360.08 69.368.2 53.267.9
t-test 0.043 0.011 0.216 0.078 0.399 0.020 0.360 0.361 8
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Figure4|PlottedfeaturesobtainedfromS1L5pyramidalneuronsthroughvoltagerecordingsinna¨ıveconditions(#)orunderthepresenceofthe
.
TRPM8channelblockerBCTC15mM( ).Theextractedfeaturesareobtainedfromdifferenttypesofstimulationprotocolsperformedoncontrol
(black)andundertheeffectofmenthol250mM(red).Firingpatternfeaturesfromsuccessiveincrementalcurrentsquarepulsesareplottedandcompared
(A).LastAPanalysis(A1)showslittleornodifferenceontheeffectproducedbymenthol250mM(red),whetherifitismeasuredintheabsence(#;n5
.
9)orinthepresenceofBCTC15mM( ;n58)(average6S.E.).AsimilarsituationcanbeobservedintheAPdroprateslopemeasured(A2;average6
S.E.;*p,0.05,***p,0.005,estimatedbyStudent’sttest).Parametersobtainedfromincrementalcurrentrampstimulationareplotted(B).Theeffect
.
producedbymenthol250mM(red)onthethreshold(B,left)wasnotblockedbyBCTC15mM( ;n58Average6S.E.*p,0.05,estimatedby
Student’sttest).Ontheactionpotentialamplitudedroprate(B,right)theeffectofmenthol250mM(red)canbeobservedinna¨ıveconditions(#;n59)
.
butstopsbeingstatisticallysignificantinthepresenceofBCTC15mM( ;n58)(average6S.E.;***p,0.005,estimatedbyStudent’sttest).The
parametersthatdescribetheactionpotentialwave-formareplotted(C)andcompared.Thelackofeffectproducedbymenthol
(red)ontheAPamplitude(left)andonthewidth(right)persistsinna¨ıveconditions(#;n59)andinthepresenceofBCTC(.;n58;Average6S.E.;
estimatedbyStudent’sttest).However,theblockerseemstohavesomeeffectonthewidth.TheinputresistancevalueswereextractedfromI/V
analysisandplotted(D)forvoltageatpeak(left)andatsteadystate(right).Foreachcasetheresistanceappearstoremaininvariantforallconditions
(estimatedbyStudent’sttest).
Based on the results obtained on the TRPM8 KO animals, our mechanisms.Thissuggeststhepresenceofanadditionalindepend-
studyshowsthattheactionofmentholandicilinonneuralactivity entmechanismofaction.
is independent of their effect on TRPM8. Mechanisms of action TheeffectsofAISappeartoinvolvevoltageandtiming-depend-
proposed by other authors include enhancement of the currents ent modulation of the sodium currents responsible for the gen-
generated by low concentrations of GABA , direct activation of eration of APs. Previous work on the peripheral nervous system
A
the GABA receptor3,23 and longer-term neuroprotective effects (PNS)hasshownthatsomeAISpresentanalgesicpropertieswhen
A
(decreasedoxidationandapoptosis,decreasedconcentrationofthe applied topically. In the case of menthol3, the mechanism of the
cytokines IL-1b and TNF-a)32,33. However, our study shows that analgesiahasbeenrelatedtomodulationofsodiumcurrents3,34,35,36
mentholandicilinareeffectiveevenwhentheGABAAreceptoris and calcium currents. When superfused with menthol3 dorsal
blockedbygabazine.ItalsoshowsthattheeffectsofAISareimme- neurons presented a similar effect on their firing pattern in a
diate, and therefore independent of longer-term neuroprotective dose-dependent manner. Our results suggest that the same or a
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Table3|AnalyzedfeaturesoftheeffectofdifferentTRPM8ligandsintheelectrophysologicalpropertiesinS1L5pyramidalneuronsin
TRPM8KOandWTmice
APDRp(mV/pA) Lp#AP(nmbr) APDRr(mV/pA) Thres(mV) APAmp(mV) APWidth(ms) IRpeak(MV) IRs.s.(MV) N
wtCtrl 20.0860.02 47.062.6 22.463.0 240.561.0 69.062.4 1.3660.07 116.4611.5 94.8612.6
wtMntl 21.8361.16 21.767.5 233.6610.6 234.161.6 65.262.6 1.4460.07 110.166.4 93.866.7
t-test 0.096 0.007 0.014 0.003 0.035 0.047 0.304 0.469 7
KOCtrl 20.0960.08 49.962.4 210.865.4 240.261.5 68.863.5 1.2660.09 98.4611.2 77.869.7
KOMntl 21.8060.56 19.767.2 255.4616.6 235.861.6 65.062.7 1.3660.14 96.9615.7 82.8614.2
t-test 0.008 0.002 0.018 0.004 0.135 0.101 0.429 0.270 7
6
wtCtrl 20.1760.02 48.463.0 219.563.9 242.960.9 76.162.1 1.0160.05 71.464.5 54.963.5
wtIcln 21.0760.37 38.866.8 227.163.6 241.360.9 76.161.8 1.0260.05 62.566.7 49.965.4
t-test 0.022 0.111 0.141 0.102 0.495 0.399 0.019 0.092 8
KOCtrl 20.2760.04 46.361.6 221.364.7 241.760.8 73.862.2 1.0360.04 74.267.3 60.465.5
KOIcln 20.5360.09 43.064.9 225.165.7 241.561.3 73.362.2 1.0560.10 61.165.4 50.864.2
t-test 0.008 0.271 0.124 0.369 0.364 0.185 0.016 0.025 7
similar mechanism is present in the brain. This mechanism Anagentthatselectivelydampenedneuralactivityinhighlydepo-
appears to be independent of TRP or GABA receptors. larized neurons could have clinical applications. Excitotoxicity
A
Given that several different AIS produce qualitatively similar (deathordamagetoneuronsduetoexcessivestimulation)isassoc-
effects,wepredictthataswellasbindingtothespecificTRPchannel iatedwithabroadrangeofconditionsincludingspinalcordinjury,
withwhichtheyareusuallyassociated(e.g.TRPM8formentholand stroke,traumaticbraininjury,andabroadrangeofneurodegenera-
icilin)thesecompoundsactonothersodium/calciumchannelsinthe tive diseases. Our own results combined with results from other
same superfamily of ion channels, perhaps by extending their groupsmentionedbefore21,22,23,31provideapreliminaryproofofcon-
physiological refractory period. If this is true, AIS modulate the ceptthatseveralAISdisplaythedesiredproperties.AIS’longhistory
positive feedback loop through which sodium currents depolarize ofsafeuseinfoodandtraditionalmedicineprovidesanadditional
the cell, and generate APs affecting the recovery time of these favourableindicationforclinicalapplications.Finally,thefactthat
currents. severaldifferentAIShavequalitativelysimilareffectsonneuralactiv-
Figure5|VoltagerecordingsfromasingleL5pyramidalneuronofaC57BL6/JTRPM8KOmouseprimarysomatosensorycortex.Theresponse
correspondstodifferentcurrentstimuliincontrol(left),menthol250mM(red,center)andwashout(right)conditions.Voltagetracesofsuprathreshold
squarepulsestimuli(A)withlowcurrent(toprow)andhighcurrentstimulation(bottomrow)inallthreeconditions.Firingpatternisstronglyaffected
bythepresenceofmenthol250mM(A,red,center)especiallyathighdepolarizationlevels.Firingbehaviourispartlyrecoveredafterwashout(A,right).
Voltageresponsetoanincreasinglydepolarizingramp(B)fromsubthresholdtosuprathresholdvaluesshowadifferencebetweencontrol(left)and
menthol250mM(red,center)conditions.Firingpatternispartlyrecoveredonthewashout(right).VoltagetracescorrespondingtheAPwave-form
(C)andtoI/VAnalysis(D)shownodifferencebetweencontrol(left)andmenthol250mM(red,center)conditions.
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Figure6|PlottedfeaturesobtainedfromS1L5pyramidalneuronsthroughvoltagerecordingsinwildtypeanimals(#)orinaTRPM8KOstrain(.).
Theextractedfeaturesareobtainedfromdifferenttypesofstimulationprotocolsperformedoncontrol(black)andundertheeffectofmenthol250mM
(red).Firingpatternfeaturesfromsuccessiveincrementalcurrentsquarepulsesareplottedandcompared(A).LastAPanalysis(A1)showslittleorno
differenceontheeffectproducedbymenthol250mM(red),whetherifitismeasuredinwildtype(#;n57)orinTRPM8KOanimals
.
( ;n57)(average6S.E.).SimilarsituationcanbeobservedintheAPdroprateslopemeasured(A2,average6S.E.;*p,0.05,***p,0.005,estimated
byStudent’sttest).Parametersobtainedfromincrementalcurrentrampstimulationareplotted(B).Theeffectproducedbymenthol250mM(red)on
thethreshold(B,left)andontheactionpotentialamplitudedroprate(B,right)ispresentwhetherobtainedfromwt(#;n57)orTRPM8KO(.;n5
7)animals(average6S.E.;*p,0.05,***p,0.005,estimatedbyStudent’sttest).Theparametersthatdescribetheactionpotentialwave-formareplotted
(C)andcompared.Thelackofeffectproducedbymenthol(red)ontheAPamplitude(C,left)andonthewidth(C,right)persistsinwt(#;n57)andin
.
TRPM8KOanimals( ;n57)(average6S.E.;estimatedbyStudent’sttest).TheinputresistancevalueswereextractedfromI/Vanalysisandplotted
(D)forvoltageatpeak(D,left)andatsteadystate(D,right).Foreachcasetheresistanceappearstoremaininvariantforallconditions
(estimatedbyStudent’sttest).
itysuggeststhatitmaybepossibletodesignsyntheticspiceanalogues effectiveness,willrequirestudiesofoptimaldosageandevaluationof
with pharmacological properties superior to those of their natural alternativemodesofadministration.
counterparts. Finally, more work is needed to understand the mechanism or
Inourstudy,AISwereadministeredviaperfusion,providingno mechanismsthroughwhichAISmodulateneuralactivity.Itmight
indication of whether AIS can cross the blood-brain barrier. bepossiblethattheseAISinteractdirectlywiththesodiumchannels
However, Zhang et al23 report that menthol has an anti-epileptic thatareresponsibleforgeneratingAPs,oritmightbethatthesizeof
effect,notonlyinculturebutalsowhenadministeredthroughintra- thesemoleculesandtheirlipophilicityaffectthelipidbilayerofneu-
peritoneal(i.p)injection.Similarly,DeAlmeidaetal22reportthati.p. ronsinawaythatdisruptstheirinactivationtau.Weinferthatthe
injection of AIS (linalool, TRPM8 ligand) present in the leaves of analgesiaeffectalreadyobservedintheliteratureonthePNSandthe
Cissussicyoidescaninhibitepilepticactivity.Thisisapparentevid- antiepilepticeffecttestedwithsomeoftheseAISmightbetwosides
encethatatleastsomeAIScancrossthebrainbarrierbyexertinga ofthesamemechanism.
similareffecttotheoneobservedinthepresentstudy,andforthose
AIStested,thattheyarenottoxicininvivoconditions21,22,23,33.
Methods
More research is needed to study whether AIS are effective in
Spices.Theeffectsoftheactiveingredientsoffivenaturallyoccurringspices
modelsofdiseasethataremorerealisticthantheadmittedlyartificial
(capsaicin-redchili;eugenol-cloves;cinnamaldehyde-cinnamon;menthol-mint;
(invitro)modelswehaveusedinthisstudy.Proofofpossibleclinical allylisothiocianate-wasabi;radish;andmustard)andoneartificialspiceanalog
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Figure7|Mentholpreventsseizure-likeburstsintheneocortex.Voltagerecordingsoflayer5pyramidalneuronsofmouseprimarysomatosensory
cortexwhileapplyinggabazinethroughincreasingconcentrationsteps(A).ElicitedburstsincreaseindurationandthenumberofAPselicitedasthe
concentrationofgabazinebecomeshigher(C).Theincreasingconcentrationsstepsofgabazineprotocolarerepeatedwhilepermanentlyapplying
menthol250mM(red).Inordertoobtainburstshighergabazineisrequired(B)andthedurationaswellasthenumberofAPseliciteddecrease(D).
(icilin)wereinvestigated.Capsaicin4andeugenol9bindtoTRPV1,menthol15and functionoftime.Inputresistanceatsub-thresholdvoltageischaracterizedby
icilin17bindtoTRPM8,andcinnamaldehydeandallylisothiocyanate(AITC)bindto stimulatingthecellwithaninitialcurrentsufficienttogenerateahyperpolarized
TRPA120.Toimprovesolubility,thecompoundsweredissolvedindimethylsulfoxide voltagepulse,followedbyasubsequentsetofsquarecurrentpulseswithstepwise
(DMSO)toformastocksolution,whichdoesnotaffectelectrophysiological increasingcurrent.Bymeasuringthevoltageatthebeginningandendofeachpulse
properties37. (peakandsteadystatevoltage),andplottingitagainstthecorrespondingvaluesof
Capsaicin,icilin,eugenol,mentholandcinnamaldehydewereobtainedfromSigma currentinjectedwecancalculatetheinputresistancesvalues.Thetimedependencyof
-Aldrich.AITCwasobtainedfromSpectrumChemicalMFG.Corp. cellbehaviouratabovethresholdvoltagesischaracterizedintermsofparameters
describingchangesintheamplitudeoftheAPsgeneratedbyadepolarizedcell
CorticalPreparations.Parasagittalcorticalpreparations(300mmthick)were whenstimulatedwithasequenceofsquarecurrentpulsesorrateofdropin
obtainedfromtheprimarysomatosensorycortexofwildtype(C57BL6/J)micepost- amplitudeofAPs,andbyadditionalparametersdescribingthelastAP
natal13–18days.AllprocedureswereconductedinconformitywiththeSwiss producedbyeachburst(overshoot,sequencenumber,width,timeafterbeginningof
WelfareActandtheSwissNationalInstitutionalGuidelinesonAnimal stimulation).
Experimentationfortheethicaluseofanimals.TheprojectwasapprovedbytheSwiss TheseparameterswereusedtocharacterizetheeffectofsixAIS(capsaicinat
CantonalVeterinaryOfficefollowingitsethicalreviewbytheStateCommitteefor 25mM,eugenolat200mM,mentholat250mM,icilinat50mM,cinnamaldehydeat
AnimalExperimentation.Allcorticalpreparationswerecutinice-coldaCSF 300mMandAITCat500mM),ontheelectricalbehaviouroflayer5pyramidalcells
(artificialcerebro-spinalfluid)withlowCa21andhighMg21.Corticalpreparations inparasagittalslicesfromthesomatosensorycortex.Atthebeginningofeach
werethentransferredto34uCfor20mininnormalaCSFandkeptatroom experiment,theslicewasperfusedwithaCSFforatleast15min.Afterafirst
temperature(20–22uC)beforethestartofrecording.NormalandcuttingaCSFwere administrationofthestimulationprotocol,theslicewasperfusedforafurther
continuouslybubbledwithamixtureof95%O and5%CO.ThecuttingaCSF 15minswithasolutionofasingleAIS,enrichedwithdimethylsulfoxide(DMSO)in
2 2
contained(mM):10MgCl,0.5CaCl,125NaCl,25glucose,25NaHCO,2.5KCl, aCSFatavol/volratioof151000.Thestimulationandrecordingprotocolswere
2 2 3
1.25NaHPO.ThenormalaCSFcontained(mM):2CaCl,1MgCl,125NaCl,25 administeredasecondtime,duringcontinuousperfusionwithAIS.Theslicewasthen
2 4 2 2
glucose,25NaHCO,2.5KCl,1.25NaHPO.BothhadapHof7.3.During perfusedwithcleanaCSFforafurther15minutes(wash-outperiod)andtheprotocol
3 2 4
recording,corticalpreparationswereconstantlyperfusedwithaCSFat2ml/minat wasadministeredforathirdtime(onlyforcellsthatwerepresentingaproperseal).In
33uC(61uC).AllchemicalcompoundswereobtainedfromSigma–Aldrich. betweenadministrationsoftheprotocol,therestingmembranepotentialwas
maintainedat270mV.
Characterizingspiceeffectsonsingleneurons.Characterizationofsingleneuron Duringthestudy,wecollecteddatafromatotalofn587S1L5pyramidalneurons
activitywasbasedonastandardizedcurrentinjectionandrecordingprotocol (Table1).Asanegativecontrol,wealsoadministeredtheprotocolonslicesperfused
developedinourlaboratory38.Theprotocolrecords50parameterscharacterizingthe withaCSFandDMSOwithoutthepresenceofAIS.Theresultingdatawerecompared
responseofthecelltoastandardizedbatteryof15electrophysiologicalstimuli. withthebaselinedataforslicesperfusedwithpureaCSF.Asasecond,positivecontrol
Individualcellsareidentifiedopticallyandpatchedinwholecell,currentclampmode eachprocedurewasrepeatedinglutamatergicneuronsfromlateralamygdalaslices.
usingamulti-patchset-up.Administrationofthecompleteprotocoltakes
approximately10minutes.Herewefocusedonasubsetoftheparametersgenerated ProbingtheinvolvementofTRPchannels.Weused4-(3-chloro-2-pyridinyl)-N-
bytheprotocolasdescribedingreaterdetailbelow. [4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide(BCTCat15mM)toblock
ParametersforActionPotentials(AP)aremeasuredusingfastsampling(20kHz) TRPM8incorticalslices.Thechoiceoftheblockerwasmotivatedbyitshigh
ofasingleAP,evokedbyasinglesquarepulseofcurrent.Thevoltageresponseofthe bindingspecificity14.BCTCwasdissolvedinDMSOtocreateastocksolution,
cellischaracterizedbygraduallyrampinguptheinputcurrent,andrecording whichwasaddedtotheaCSF.Sliceswereperfusedfor10minuteswithBCTC1
changesinvoltage,includingtheAPsgeneratedwhenthevoltagepassesfromsub-to aCSF.Wethenrepeatedthecurrentinjectionandrecordingprotocolusedinour
abovethresholdvalues.Theamplitudeoftheelicitedactionpotentialsisplottedasa previoussingleneuronstudies(controlcondition).Afteradditionofeither
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Figure8|Voltagerecordingsintheabsence(control)orinthepresenceofthedifferentAISwereobtainedwhileGabazinewasappliedinan
incrementalmannerthrough5minsteps.APburstswerespontaneouslygenerated(A).APburstswereanalyzedandquantifiedfortwodifferent
concentrationsofGabazine(10mMand20mM),byarbitrarilyplacingadepolarizationlimitat240mV(B).Datawasplottedastotalnumberofevents
(C),andtotaldurationofthedepolarizedphase(D).
mentholoricilinwerepeatedtheprotocol(experimentalcondition).Finally,we burstingbehaviourreminiscentofseizure.Usingthismodel,weinjectedaconstant
comparedtheeffectsofAISinslicesperfusedwithBCTCwiththeeffectsonna¨ıve currentintotheneuronsinwholecellcurrentclampingmode,settingthemembrane
slices. voltageto260mV-asubthresholdvalue,andmeasuringtheirresponsetogabazine
Tovalidateourresults,werepeatedthestudyusingaTRPM8KOmouse intheabsenceandpresenceofindividualAIS.Atthebeginningofeachexperiment
(Trpm8tm1Apat/J)inaB6;129S1(FVB)background13.Theresultswerecomparedwith theslicewasperfusedfortenminuteswithasingleAISdilutedinaCSF,asdescribed
WTlittermatesofthesamestrainwithsameageandsex.BCTCwasobtainedfrom previously.WethenaddedgabazinetotheaCSF,startingatadoseof1mMand
BioTrends. increasingthedoseindiscretestepsevery5minutesuptoamaximumdoseof
60.0mM.Finally,theslicewasperfusedwithpureaCSF(nogabazine,noAIS)fora
Testingforanti-epilepticeffects.Totestforputativeanti-epilepticeffectsofAIS,we wash-outperiodofatleast10minutes.Cellfiringbehaviourwasrecordedforthe
usedaninvitromodelofepilepsyinwhichcorticalslicesareperfusedwithstepwise wholedurationoftheexperiment.
increasingdosesofgabazine(SR-95531),aGABAAreceptorantagonistthatblocks Datawerecollectedfrom26S1L5pyramidalneurons.Asanegativecontrol,we
theactionofinhibitoryinterneurons,up-regulatingsliceactivityandinducing repeatedtheprotocolonslicesperfusedwithaCSFandDMSOwithoutthepresence
Table4|Totalnumberofeventsin5mininthepresenceofthedifferentAIS,with10 mMor20 mMofGabazineAvg.6S.E.
N Gab10mM t-test Gab20mM t-test N
Control 3.7560.48 4.0060.58 4
Capsaicin25mM 3.4060.25 0.273 1.260.49 0.006 5
Eugenol200mM 0.060.0 0.003 0.060.0 0.006 3
Menthol250mM 1.5060.5 0.009 1.2560.25 0.002 4
Icilin50mM 0.8060.49 0.002 1.4060.40 0.004 3
AITC500mM 6.0061.15 0.05 50.0062.31 0.00002 3
Cinnamaldehyde300mM 23.7569.44 0.039 16.5067.42 0.107 4
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