Decreased Subcortical Cholinergic Arousal in Focal Seizures

13 Pages·2015·3.05 MB·English

by  Joshua E. Motelow

Article
Decreased Subcortical Cholinergic Arousal in Focal
Seizures
Highlights Authors
d SubcorticalarousalnucleishowdecreasedfMRIsignalsin JoshuaE.Motelow,WeiLi,...,
focalseizures FahmeedHyder,HalBlumenfeld
d Seizuressuppresscholinergicneuronsinthebrainstemand Correspondence
basalforebrain
[email protected]
d Amperometryrevealsdecreasedacetylcholineinthecortex
In Brief
andthalamus
Intenseincreasedfiringofneuronsis
d Mechanismoflossofconsciousnessinfocalseizures commonlythoughttodisruptbrain
resemblesslow-wavesleep functioninepilepsy.Motelowetal.
demonstrateinsteadthatdecreased
activityinsubcorticalarousalsystems
leadstodepressedcorticalfunctionand
impairedconsciousnessinfocalseizures.
Motelowetal.,2015,Neuron85,561–572
February4,2015ª2015ElsevierInc.
http://dx.doi.org/10.1016/j.neuron.2014.12.058
Neuron
Article
Decreased Subcortical Cholinergic
Arousal in Focal Seizures
JoshuaE.Motelow,1WeiLi,1,2QiongZhan,1,3AshtM.Mishra,1RobertN.S.Sachdev,4GeoffreyLiu,1
AbhijeetGummadavelli,1ZainaZayyad,1HyunSeungLee,1VictoriaChu,1JohnP.Andrews,1DarioJ.Englot,8
PeterHerman,6,7BasavarajuG.Sanganahalli,6,7FahmeedHyder,6,7andHalBlumenfeld1,4,5,7,*
1DepartmentofNeurology,YaleUniversitySchoolofMedicine,NewHaven,CT06520,USA
2DepartmentofNeurosurgery,JinlingHospital,SchoolofMedicine,NanjingUniversity,Nanjing,210002,JiangsuProvince,People’sRepublic
ofChina
3DepartmentofNeurology,XiangyaHospital,CentralSouthUniversity,Changsha,Hunan410008,China
4DepartmentofNeurobiology
5DepartmentofNeurosurgery
6DepartmentofDiagnosticRadiology
7CoreCenterforQuantitativeNeurosciencewithMagneticResonance(QNMR)
YaleUniversitySchoolofMedicine,NewHaven,CT06520,USA
8DepartmentofNeurologicalSurgery,UniversityofCalifornia,SanFrancisco,SanFrancisco,CA94143,USA
*Correspondence:[email protected]
http://dx.doi.org/10.1016/j.neuron.2014.12.058
SUMMARY lobe seizures, the cerebral cortex exhibits widespread slow-
waveactivityinterpretedbysomeasseizurepropagation(Lieb
Impaired consciousness in temporal lobe seizures etal.,1991).Instead,weproposethatthesecorticalslowoscil-
has a major negative impact on quality of life. The lationsandthecoincidentreductionincerebralbloodflowrepre-
prevailingviewholds thatthisdisorderimpairscon- sentdepressedcorticalactivity(Blumenfeldetal.,2004a,2004b;
sciousnessbyseizurespreadtothebilateraltempo- Englotetal.,2008,2009,2010).Iftrue,thesubcorticalarousal
ral lobes. We propose instead that seizures invade systems should be depressed (Blumenfeld, 2012; Norden and
Blumenfeld, 2002)as in other states of unconsciousness such
subcortical regions and depress arousal, causing
asdeepsleep(Steriadeetal.,1993b)orcoma(Young,2000).
impairment through decreases rather than through
Reframing impaired consciousness in temporal lobe sei-
increases in activity. Using functional magnetic re-
zures as a deficit in subcortical arousal leads to several pre-
sonance imaging in a rodent model, we found
dictions (Figure 1). Increased activity is expected in limbic
increased activity in regions known to depress cor-
archicortical structures such as the hippocampus but also in
tical function, including lateral septum and anterior connected subcortical inhibitory regions such as the lateral
hypothalamus. Importantly, we found suppression septal nuclei and anterior hypothalamus, known to project to
of intralaminar thalamic and brainstem arousal sys- the subcortical arousal systems (McGinty and Szymusiak,
tems and suppression of the cortex. At a cellular 2001; Mesulam and Mufson, 1984; Varoqueaux and Poulain,
level, we found reduced firing of identified cholin- 1999). Key subcortical arousal regions, such as the brainstem
ergic neurons in the brainstem pedunculopontine pedunculopontinetegmentalnucleus(PPT),thalamicintralami-
nar nuclei (Glenn and Steriade, 1982; Heckers et al., 1992),
tegmental nucleus and basal forebrain. Finally, we
and the basal forebrain cholinergic nuclei (Mesulam et al.,
used enzyme-based amperometry to demonstrate
1983b)areexpectedtoshowreducedactivity.Inaddition,the
reduced cholinergic neurotransmission in both cor-
releaseofmodulatoryarousalneurotransmitterssuchasacetyl-
tex and thalamus. Decreased subcortical arousal is
choline should decrease in both cortex (Marrosu et al., 1995)
a critical mechanism for loss of consciousness in
and thalamus (Williams et al., 1994). We previously developed
focaltemporallobeseizures. a rodent model that resembles human temporal lobe seizures
in terms of electrophysiology and behavioral deficits (Englot
etal.,2008,2009).However,toourknowledge,littleworkhas
INTRODUCTION been done to investigate decreased subcortical arousal as a
mechanismofimpairmentinepilepsy.
Why do patients lose consciousness during temporal lobe In the present study, we use a combination of high-field
seizures?Thetemporallobesarenottraditionallyapartofthe blood-oxygen-level-dependent (BOLD) functional magnetic re-
consciousness system (Blumenfeld, 2012; Saper et al., 2005); sonanceimaging(fMRI),directelectrophysiologicalrecordings,
patient HM was conscious without both hippocampi (Scoville and amperometry-based neurotransmitter measurements to
andMilner,1957).Yetinfocaltemporallobeseizures,patients investigate the modulation of subcortical arousal during limbic
often become unresponsive and demonstrate automaton-like seizures on multiple levels. We found that depressed cortical
behavior(Escuetaetal.,1977;Penfield,1952).Duringtemporal function—evidencedbycorticalslowoscillationsanddecreased
Neuron85,561–572,February4,2015ª2015ElsevierInc. 561
FrontalCtx Glu HC
FFFF
Thalamus,
ACh,others Basal Hypothal,
Forebrain LS
GABBAA Excitation
Inhibition
ACh,others PPT,other DecreasedExcitation
subcort
arousal
Figure1. NetworkInhibitionHypothesisandPredictions
Prediction1:Seizureactivityinthehippocampus(HC)propagates(viafimbria-
fornix,FF,andotherpathways)toincreaseactivityininhibitoryandsleep-
promotingregionssuchastheanteriorhypothalamus(Hypothal)andlateral
septum(LS);asaconsequence,subcorticalarousalsystemsincludingthe
thalamus,basalforebrain,PPT,andothersubcorticalarousalsystemsshow
depressedactivity.Prediction2:Firingofidentifiedarousalneurons,suchas
cholinergicneuronsinPPTandbasalforebrain,decreasesduringseizures.
Figure 2. Hippocampal, Cortical and Subcortical BOLD fMRI
Prediction 3: Release of arousal neurotransmitters including acetylcholine
ChangesduringLimbicSeizures
(ACh)isdecreasedinboththalamusandcortex(Ctx).Thisinturnleadsto
T-mapofictalchangesduringpartialseizures(from30spre-seizurebaseline)
decreasedcorticalarousalandtoslow-wavesinthecortex.
revealscomplicatednetworkofchanges.Widespreadcorticaldecreasesare
accompaniedbymixedsubcorticalincreasesanddecreases.Increasesare
seeninknownareasofseizurepropagationsuchasthehippocampus(HC)
fMRIsignals—isaccompaniedbyincreasedactivityinthehippo- andlateralseptumaswellasinsleep-promotingregionssuchastheanterior
campus, anterior hypothalamus, and lateral septum. fMRI sig- hypothalamus(AntHyp).Decreasesareseeninthecortex,mostprominently
nalsweredecreasedintheintralaminarthalamusandmidbrain in lateral and ventral orbital frontal cortex (LO/VO) and in medial regions
tegmentum,guidingustoperformjuxtacellularrecordingsfrom including cingulate and retrosplenial cortex. Decreases are also seen in
arousalpromotingregionssuchasthethalamicintralaminarnucleiincluding
the mesopontine cholinergic nuclei, which provide cholinergic
centrolateralnucleus(CL),aswellasinthemidbraintegmentum(MT).The
excitation of the thalamus (McCormick, 1992; Mesulam et al., arrowheadsatAP(cid:1)3.4mmsignifythehippocampalelectrodeartifact.Warm
1983b).WefoundsuppressedfiringinPPTcholinergicneurons. colorsrepresentfMRIincreases,andcoolcolorsdecreases,superimposedon
Further exploring the cholinergic arousal system, we investi- coronalanatomicalimagesfromthetemplateanimal.APcoordinatesinmil-
gatedneuronsinthecholinergicbasalforebrain,whichinnervate limetersrelativetobregma(PaxinosandWatson,1998).10animals,withFDR
the neocortex (Mesulam et al., 1983a; Rye et al., 1984), and correctedthresholdp<0.05.SeealsoFigureS1.
foundthemsuppressedaswell.Finally,weshowedthatcholine,
asaproxyforacetylcholine,isdecreasedinboththecortexand zures(Figures2and3).BOLDincreaseswerefoundinthehippo-
thalamus during limbic seizures. Decreased acetylcholine has campus,hypothalamus,andseptumwhiledecreaseswereseen
longbeenassociatedwithcorticalslowoscillations,aninhibited inthecortex,intralaminarthalamus,andmidbraintegmentum.
thalamus,andasuppressedbasalforebraininslow-wavesleep Guided by the fMRI t-map results (Figure 2), we targeted six
(Marrosu et al., 1995; Williams et al., 1994). These data pro- regionsofinterest(ROI)(Figure3A)toinvestigatechangesover
videdirectevidenceofseizureactivitysuppressingsubcortical time. Three of the ROIs, namely hippocampus (HC), septum,
arousalasamechanismofdepressedcorticothalamicnetwork andanteriorhypothalamus(AntHyp),showedprominentBOLD
function. fMRI increases followed by postictal decline and eventually a
returntobaseline(Figure3B).Theseregionsallshowincreased
RESULTS multiunit activity ictally with rapid decrease postictally (Fig-
ure S1), suggesting that the slow decay of the BOLD signal
fMRI intheseregionspostictallymayrepresentperfusiondecreasing
HighfieldBOLDfMRIrevealedanetworkofcorticalandsubcor- more slowly than neural activity (Hyder et al., 2010). All three
tical changesconsistent withdecreased subcortical arousal in regions showed significant increases during seizures com-
limbicseizures.WeconductedfMRImeasurementsduringpar- pared to baseline (Figure 3C) (comparing seizure to base-
tial limbic seizures (10 animals, 34 seizures, mean duration ± line: HC +3.63% ± 0.40%, Septum +3.04% ± 0.53%; Ant
SEM70.72±4.01s),inducedbybriefhippocampalstimulation Hyp+2.06%±0.28%;one-samplettestsHolm-Bonferronicor-
(Englot et al., 2008, 2009). A quadrature coil design (Hyde rected,p<0.05).Thehypothalamusplaysakeyroleinpromot-
etal.,1987)improvedthedetectionpowerofourfMRIrecord- ingslow-wavesleep(Saperetal.,2005),andstimulationofboth
ingsincriticalventralsubcorticalareasnotvisualizedpreviously thelateralseptum(Englotetal.,2009)andanteriorhypothalamus
duringseizures(Englotetal.,2008,2009). (StermanandClemente,1962)causescorticalslowoscillations
We found decreased cortical fMRI activity paired with both resemblingdeepsleep.Thesechangesarethereforeconsistent
subcortical increases and decreases during partial limbic sei- with a model in which loss of consciousness during partial
562 Neuron85,561–572,February4,2015ª2015ElsevierInc.
tegmentum all showed significant fMRI decreases during sei-
zures compared to baseline (Figure 3C) (comparing seizure
to baseline: LO (cid:1)3.65% ± 0.61%; CL (cid:1)0.93% ± 0.30%; MT
(cid:1)1.07% ± (cid:1)0.27%; one-sample t tests Holm-Bonferroni cor-
rected, p < 0.05). The brainstem tegmentum is comprised of
arousal nuclei, including the major source of cholinergic input
for the intralaminar thalamus (Hallanger et al., 1987; Mesulam
et al., 1983b). This circuit is vital for promoting the excitatory
actionsofthethalamusoncortexandformaintainingthalamic
neuronsintheirregularfiringmode(GlennandSteriade,1982;
McCormick, 1992). The thalamus and midbrain tegmentum
havelongbeenimplicatedinarousal,workingbothcooperatively
andindependently (GlennandSteriade,1982;Hallangeretal.,
1987;Mesulametal.,1983b;MoruzziandMagoun,1949;Poulet
etal.,2012;Steriadeetal.,1993a).
PPTJuxtacellularRecordings
Our BOLD data showed decreased signal across both the
midbrain tegmentum andintralaminar thalamus (Figures2and
3).Thearousal-promotingbrainstemcholinergicsystemlocated
in the brainstem tegmentum, centered in the PPT, provides a
physiologicalbridgebetweenthetwoanatomicalregions(Ster-
iadeetal.,1993a).Acetylcholineoriginatingfromthebrainstem
has a profound direct effect on the thalamus (McCormick,
1992) and therefore serves as an indirect vehicle of cortical
arousal (Moruzzi and Magoun, 1949; Steriade et al., 1993a).
The PPT is a heterogeneous nucleus although the non-cholin-
ergicneuronsalsohaveaputativearousalroleviatheforebrain
cholinergicsystem(Steriadeetal.,1993a).Todeterminewhether
the brainstem cholinergic system is truly suppressed during
limbic seizures, we conducted juxtacellular recordings in the
PPT. Locations of all identified neurons recorded in the PPT
Figure3. BOLDROITimeCoursesRevealIncreasesandDecreases andperi-PPTregionareinFigureS2.
duringSeizuresandEventualReturntoBaseline Arepresentativerecordingfromacholinergicneuronisshown
(A)ExampleROIonanatomicalbrainimages.
(Figure 4). The neuron fired regularly prior to seizure initiation
(B)MeanROItimecourses(±SEM)fordata30spriortoseizureonset,seizure
(Figure4A,Baseline).Duringtheseizureinthehippocampus(Fig-
time course scaled to mean seizure duration, and unscaled postictal time
ure4A,Seizure),thecorticalmultiunitactivity(MUA)convertedto
coursealignedtoseizureend.
(C)MeanictalBOLDchangeisincreasedinlimbicandsleep-promotingre- UpandDownstates(Steriadeetal.,1993b)ofalternatingfiring
gionsanddecreasedincorticalandarousalregions.ErrorbarsareSEM.All andquiescence,whilethecorticallocalfieldpotential(LFP)con-
changesduringseizuresweresignificantlydifferentfrombaseline(one-sample verted to prominent slow oscillations, as described previously
ttestsHolm-Bonferronicorrected,p<0.05).Dataarefromsameanimalsand (Englot et al., 2008). At the same time, the cholinergic neuron
seizuresasinFigure2.AntHyp,anteriorhypothalamus;HC,hippocampus;
markedly decreased its firing almost immediately after the
LO,lateralorbitalfrontalcortex;CL,centrallateral(intralaminar)thalamus;MT,
seizurebegins.Inthepostictalperiod(Figure4A,Postictal),the
midbraintegmentum;Septum,lateralseptalnuclei.SeealsoFigureS1.
corticalLFPwasstilldominatedbylow-frequencyoscillations,
and the cholinergic neuron remained depressed, but its firing
seizuresiscausedbyincreasedactivityininhibitoryprojection ratelaterbegantorecover.Afterafewminutes,theanimalrecov-
areas(Figure1). eredbacktobaseline(Figure4A,Recovery).ThecorticalLFPwas
Three ROIs were chosen to investigate BOLD decreases againdominatedbyhigherfrequencyactivityresemblingwake-
(Figure 3A). We have previously reported BOLD decreases in fulness, and the cholinergic neuron again fired regularly. The
the lateral orbital frontal cortex (LO), which is consistent with neuronrecordedhere,whichco-stainedforcholineacetyltrans-
decreased metabolic activity associated with ictal neocortical ferase,canbeseeninFigure4B.
slow activity (Blumenfeld et al., 2004a; Englot et al., 2008, Identified cholinergic neurons as a group displayed a con-
2009, 2010). In addition, two major subcortical arousal areas sistentanddramaticdecreaseinfiringduringseizures(seeFig-
were chosen, the intralaminar central lateral thalamus (CL) ures6Aand6B)(eightneuronsfromeightanimals;meanseizure
andmidbraintegmentum(MT).TheBOLDsignalinthesethree duration:60.34s±6.22s;comparingseizuretobaseline:mean
ROIs decreased during partial seizures, remained suppressed changeinfiringrate(cid:1)2.31Hz±0.71Hz;pairedttestHolm-Bon-
postictally,andthengraduallyreturnedtobaseline(Figure3B). ferroni corrected, p < 0.05). Although they possessed variable
The orbital frontal cortex, intralaminar thalamus, and midbrain firing rates atbaseline, all cholinergic neurons decreased their
Neuron85,561–572,February4,2015ª2015ElsevierInc. 563
Figure 4. Brainstem Cholinergic Neuron
CeasesFiringduringPartialSeizure
(A) Cholinergic neuron in PPT dramatically de-
creases firing during seizure activity. Expanded
segments of baseline, seizure, and postictal re-
cordingsfromtheboxedregionsin(A)aswellasa
recoveryperiod7minafterthestimulus.
(B)Labeledcholinergiccellrecordedin(A)stained
for Neurobiotin in left panel, choline acetyl-
transferase(ChAT)inmiddlepanel,andmergein
right panel. Scale bars are 20 mm. HC, hippo-
campus; PPT, pedunculopontine tegmental nu-
cleus;LO,lateralorbitofrontalcortex;LFP,local
fieldpotential;SUA,singleunit(juxtacellular)ac-
tivity;MUA,multiunitactivity.SeealsoFigureS2.
7.06 s; comparing seizure to baseline:
mean change in firing rate (cid:1)2.44 Hz ±
1.17 Hz; paired t test, p = 0.06), while
the bursting neurons trended toward
increased firing (Figures S4F and S4G)
(8cells;meanseizureduration63.93s±
9.00 s; comparing seizure to baseline:
mean change in firing rate +6.71 Hz ±
8.84Hz;pairedttest,p=0.47).
Inordertogetamorecompletepicture
oftheoverallregionalactivitywithinand
around the PPT, we also analyzed the
single-cell juxtacellular recordings in the
populationofneuronsinwhichtheelec-
trode tract approached the PPT but the
specificrecordedcellcouldnotberecov-
firing rates during seizures. The firing rates slowly recovered eredhistologically.Thechangeinfiringoftheseneuronsduring
duringthepostictalperiodandresumednormalfiringafteravar- seizures was significantly decreased (Figures S4H and S4I)
iableinterval.Allcholinergicneuronsfiredtonicallyandrelatively (33neurons from19animals:meanseizureduration54.73s±
slowly.Noneexhibitedburstfiring. 6.22 s; comparing seizure to baseline: mean change in firing
The non-cholinergic neurons in the PPT region are pre- rate (cid:1)3.44 Hz ± 0.72 Hz; paired t test Holm-Bonferroni cor-
dominately glutamatergic and GABAergic (Wang and Morales, rected, p < 0.05). The overall decrease in firing rate of cells in
2009).Theputativearousal-promotingroleoftheglutamatergic thisregionwasconsistentwithdecreasedBOLDsignal(Figure2)
neuronsentailsexcitingthecholinergicbasalforebrain(Steriade inthebrainstemtegmentumduringseizures.
etal.,1993a).Wedidnotdistinguishbetweenthetwocelltypes
inthisstudy,butwefoundthatunlikecholinergicneurons,which BasalForebrainJuxtacellularRecordings
consistently decreased their firing during seizures, non-cholin- WhilethecholinergicneuronsinPPTindirectlyactivatethecor-
ergic neurons showed mixed behaviors, including decreased, tex,thecholinergicneuronsofthebasalforebrainprojectdirectly
increased, or no change in firing rates. An example recording to neocortex (Mesulam et al., 1983a; Rye et al., 1984), and
fromanon-cholinergicPPTneuronisshowninFigureS3,with acetylcholinehasaprofoundeffectonstatechangesinthecor-
noappreciablechangeinfiringduringapartialseizure.Overall, tex(Kalmbachetal.,2012;MetherateandAshe,1991;Methe-
themixtureofincreased,decreased,ornochangeinfiringrate rateetal.,1992).LikePPT,thecholinergicneuronsinthebasal
of non-cholinergic neurons during the seizure period led to no forebrain are intermingled with non-cholinergic cells, although
significant change from baseline on average (see Figures 6C these neurons may have arousal promoting effects as well
and 6D) (21 neurons from 19 animals; mean seizure duration: (Freund and Meskenaite, 1992). Given the suppression of the
62.19s±5.42s;comparingseizuretobaseline:meanchange brainstem cholinergic system, the presence of cortical slow
in firing rate +1.04 Hz ± 3.45 Hz; paired t test, p = 0.76). waves,andtheincreasedactivityinslow-wavesleep-promoting
We observed that some non-cholinergic neurons began firing regionsonfMRI(Figure2),weextendedourjuxtacellularrecord-
in bursts during seizures (Figure S4), which accounts for the ingstothebasalforebrain.Locationsofallidentifiedneuronsre-
apparentriseinmeanfiringrateinthelateictalperiod(Figure6D). cordedinthebasalforebrainareinFigureS5.
Thenon-burstingneuronstrendedtowarddecreasedfiring(Fig- A representative recording from a cholinergic neuron in
ures S4D and S4E) (13 cells; mean seizure duration 61.11 s ± basalforebrainisshown(Figure5).Theneuronfirestonicallyat
564 Neuron85,561–572,February4,2015ª2015ElsevierInc.
Figure 5. Basal Forebrain Cholinergic
Neuron Decreases Firing during Partial
Seizure
(A)Cholinergicneuroninbasalforebraindramati-
cally decreases firing during seizure activity.
Expanded segments of baseline, seizure, and
postictalrecordingsfromtheboxedregionsin(A)
as well as a recovery period 4.5 min after the
stimulus.
(B)Labeledcholinergiccellrecordedin(A)stained
for Neurobiotin in left panel, choline acetyl-
transferase (ChAT) in middle panel, and merge
inrightpanel.Scalebarsare20mm.HC,hippo-
campus; BF, basal forebrain; LO, lateral orbito-
frontal cortex; LFP, local field potential; SUA,
single unit (juxtacellular) activity; MUA, multiunit
activity.SeealsoFigureS5.
cles; some neurons are correlated with
wakefulness and dysnchronous EEG
andsomearenot(Hassanietal.,2009).
AswedidwithPPTnon-cholinergicneu-
rons,wedidnotdistinguishbetweenthe
twocelltypesinthebasalforebrain.Un-
like basal forebrain cholinergic neurons,
whichconsistentlydecreasedtheirfiring
duringseizures,non-cholinergicneurons
showed mixed behaviors during sei-
zures. An example recording from a
non-cholinergic basal forebrain neuron,
which increases its firing rate, is shown
in Figure S6. Overall, the mixture of
baseline(Figure5A,Baseline),decreasesduringtheseizure(Fig- increased,decreased,ornochangeinfiringrateofnon-cholin-
ure 5A, Seizure), remains suppressed postictally (Figure 5A, ergicneuronsduringtheseizureperiodledtoanon-significant
Postictal),andeventuallyreturnstobaseline(Figure5A,Recov- decrease in firing during seizures (Figures 6G and 6H) (18
ery). As in the example shown for the PPT neuron (Figure 4), neurons from 12 animals; mean seizure duration: 86.61 s ±
the cortical LFP shows high-frequency low-voltage activity 10.16 s; comparing seizure to baseline: mean change in firing
associatedwithMUAtonicfiringatbaseline(Figure5A,Base- rate(cid:1)1.97Hz±1.71Hz;pairedttest,uncorrectedp=0.4).
line). During the seizure, the cortical LFP converts to low-fre- The temporal relationship between single neuron firing and
quencyhigh-voltageoscillationsassociatedwithUpandDown low-frequencyoscillationsinthecortexisdisplayedinFigureS7.
statesinMUA(Figure5A,Seizure).Thedepressedcorticalstate Cortical LFP 1–4 Hz power increases during seizures and de-
persists postictally (Figure 5A, Postictal) and then returns to creasesslowlypostictally(FigureS7A).Incontrast,cholinergic
its baseline state (Figure 5A, Recovery). The neuron recorded neurons in PPT (Figure S7B) and basal forebrain (Figure S7C)
here, which co-stained for choline acetyltransferase, can be decreasefiringduringseizuresandrisebacktobaselinepostic-
seeninFigure5B. tally. Non-cholinergic neurons in the PPT (Figure S7D) and
Identified cholinergic neurons in basal forebrain consistently basal forebrain (Figure S7E) had inconsistent relationship to
decreasedtheirfiringduringseizures(Figures6Eand6F)(seven corticalLFP.
neurons from six animals; mean seizure duration: 77.21 s ±
16.17 s; comparing seizure to baseline: mean change in firing CholineRecordings
rate(cid:1)4.36Hz±1.01Hz;pairedttestHolm-Bonferronicorrected, Tostudytherelationshipbetweenacetylcholinergicneurotrans-
p<0.05).Allcholinergicneuronsdecreasedfiringduringseizures. mission and ictal neocortical slowing, we measured choline
AsinPPT,non-cholinergicneuronsinthebasalforebrainare levels using high-temporal resolution amperometry (Parikh
predominantly glutamatergic and parvalbumin- and neuropep- etal.,2004).Traditionalmethodsofmeasuringneurotransmitters
tideY-containingGABAneurons(Brashearetal.,1986;Duque suchasmicrodialysissufferfromlowtemporalresolution,mak-
etal.,2007).TheGABAergicneuronsmaypromotearousalby ing detection of transient changes during seizures challenging
inhibitingcortical interneurons(Freund andMeskenaite, 1992), (Marrosuetal.,1995;Parikhetal.,2004).Enzyme-basedamper-
although both GABAergic and glutamatergic neurons have ometryallowedustomeasurecholinelevelsasaproxyforextra-
variedrelationships to thecorticalEEGand tosleep-wake cy- cellular acetylcholine with sub-second timing (Burmeister and
Neuron85,561–572,February4,2015ª2015ElsevierInc. 565
Figure6. CholinergicNeuronsinPPTandBasalForebrainDecreaseFiringduringPartialSeizureswhileFiringofNon-Cholinergicnearby
NeuronsIsMixed
(AandB)PartialseizuressuppressedfiringinPPTcholinergicneuronsduringseizures.(A)Rasterplot(eightcellsfromeightanimals)and(B)meanfiringrate
histogramdata.
(CandD)PPTnon-cholinergicneuronshaveavariablefiringrateduringseizures.Meanfiringrateduringseizures.(C)Raster(21cellsfrom19animals)and(D)
meanfiringratehistogramdata.
(EandF)Partialseizuressuppressedfiringinbasalforebraincholinergicneuronsduringseizures.(E)Raster(7cellsfrom6animals)and(F)meanfiringrate
histogramdata.
(GandH)BFnon-cholinergicneuronshaveavariablefiringrateduringseizures.(G)Raster(18cellsfrom12animals)and(H)meanfiringratehistogramdata.
Althoughselected30ssegmentsaredisplayedhere,theentiretimecoursesofmeanfiringareshowninFigureS7.Statisticswereperformedontheentireseizure
periodversusbaselineforallexperiments.Baselineis30spriortoseizureonset.Seizureisthefirst30sfollowingseizureonset.Postictalisthefirst30sfollowing
seizureend.Recoveryisthelast30sbeforeeithertheanimalwasre-anesthetizedortheneuronwaslabeledbythejuxtacellularmethod.Neuronsinrasterplots
([A],[C],[E],and[G])areorderedbasedonbaselinefiringrateforvisualization.Firingrates([B],[D],[F],and[H])arebinnedby1s.SeealsoFiguresS2–S7.
Gerhardt,2003;Parikhetal.,2004).Corticalacetylcholineorigi- cholineduringtheseizureandgradualrecoverybacktobaseline
natesprimarilyfromthebasalforebrain(Ryeetal.,1984)while postictally. Simultaneous recordings of the hippocampal and
thalamicacetylcholineoriginatesfromthebrainstemcholinergic corticalLFPshowedcorticalslowoscillationsduringtheseizure
nuclei(Hallangeretal.,1987). andpostictalperiodfollowedbynormalcorticalfastactivityafter
Wehavealreadydemonstrateddirectevidenceofsuppressed recovery(Figure7Bandlowerinsets).
firinginbrainstemandbasalforebraincholinergicneurons(Fig- Onagrouplevel,cholinedecreasedsignificantlyinthecortex
ures4,5,6A,6B,6E,and6F).Toconfirmthatacetylcholinelevels during partial seizures (Figures 8A and 8C) (ten seizures from
wereinfactsuppressedinarousal-relatedtargetareasofthese six animals; mean seizure duration 68.21 s ± 6.26 s, com-
nuclei,wemeasuredcorticalandthalamiclevelsofcholine.An paringseizuretobaseline:meancholineconcentrationchange
example choline recording from the orbital frontal cortex is (cid:1)0.086mM±0.032mM;pairedttestHolm-Bonferronicorrected,
shown(Figure7A).Cholinerecordingsintheorbitalfrontalcortex p<0.05).AsimilarpicturewasseeninCL,animportanttargetof
during limbic seizures revealed choline decreases during sei- brainstemcholinergicneuronsintheintralaminarthalamus.The
zuresthatpersistedpostictallyandeventuallyreturnedtobase- thalamic choline signal decreased during partial seizures (Fig-
line(Figure7A).Afterastimulusartifact,whichcausedincreased ures8Band8C)(sevenseizuresfromfiveanimals;meanseizure
signal in all four electrodes, the choline-oxidase-coated elec- duration53.11s±5.51s,comparingseizuretobaseline:mean
trodes showed decreased signal during the seizure. At the choline concentration change (cid:1)0.031 mM ± 0.009 mM; paired
sametime,thesentinelelectrodesreturnedtobaselinefollowing ttestHolm-Bonferronicorrected,p<0.05).Thesignaldeclined
the stimulus artifact. Subtraction of these recordings provided duringtheseizuresandthenbegantoreturntobaselinelevelsas
the self-referenced choline signal, which reveals decreased theanimalsrecoveredinbothcortex(Figure8A)andthalamus
566 Neuron85,561–572,February4,2015ª2015ElsevierInc.
Ourdatapresentaverydifferentmechanismforimpairedcor-
tical function, demonstrating that subcortical arousal circuits
showmarkedlydecreasedactivityinlimbicseizuresassociated
with a depressed cortex resembling deep sleep or coma.
Our data identify suppression of subcortical arousal systems
duringpartiallimbicseizures.Suppressedcholinergicandother
subcorticalarousalisamechanismconsistentwiththe‘‘network
inhibitionhypothesis’’(Figure1)forlossofconsciousnessduring
complexpartialtemporallobeseizures(Blumenfeld,2012;Nor-
den and Blumenfeld, 2002). We have presented data in which
apartialseizuredrivesatransitionfroman‘‘aroused’’statechar-
acterizedby‘‘fast’’corticalLFPintoa‘‘depressed’’statemarked
bycorticalslow-wavesanddecreasedfMRIsignalsinthecortex,
thalamus, and brainstem (Figures 2 and 3). Given the role of
cholinergicneuronsinprovidingexcitatoryinputtotheintralami-
nar thalamus and cortex (Steriade, 2004), we recorded from
identifiedneuronsinthebrainstemandbasalforebrain.Inagree-
ment with our hypothesis, firing of cholinergic neurons in both
nuclei was consistently suppressed (Figures 4, 5, and 6). To
studyeffectsoncholinergicoutputtocortexandthalamus,we
measuredcholinelevelsandfounddecreasesinboththeintra-
laminar thalamus and cortex during partial seizures (Figures 7
and8).Overall,thesecoordinatedchangesprovidestrongevi-
Figure7. DecreasedCorticalCholineAccompaniesCorticalSlow- denceforreducedsubcorticalarousalduringpartialseizures.
WavesduringPartialLimbicSeizure
Our data reveal a mechanism underlying loss of conscious-
(A)Amperometriccholinerecordingsinthecortexduringpartialseizure.Cyan
nessduringpartialseizuresthatmimicselementsofslow-wave
representsthecholinesignalobtainedbysubtractingmeansignalfromtwo
sentinelelectrodes(Green1,2)frommeanofthetwocholineoxidase-coated sleep. The ictal slow oscillation has similar properties to the
electrodes(Red,Ch-Ox1,2).Currentisonleftaxis,andcholineconcentration slowoscillationofslow-wavesleepandislikelyprecipitatedby
onrightaxis,obtainedbyinvitrocholinecalibrationforeachelectrode(see similar factors. Inboth slow-wave sleep and ictalslow oscilla-
alsoFigureS8). tions, low-frequency activity dominates the EEG, and Up and
(B)Simultaneousrecordingofpartialseizure.Localfieldpotential(LFP)signals
Down states are seen in the MUA. In slow-wave sleep, this is
frombothhippocampus(HC)andlateralorbitalfrontalcortex(LO)areshown.
thought to be facilitated by decreased firing of subcortical
Hippocampalseizureselicitslowoscillationsinthecortexthatpersistpos-
arousalneuronsanddecreasedarousalneuromodulatorssuch
tictallybeforerecoveringbacktobaseline(lowerinsets).
as acetylcholine in the cortex and thalamus (Marrosu et al.,
1995;McCormickandBal,1997),andourdatarevealedasimilar
(Figure8B).Thisreflectssuppressionofthecholinergicneuro- mechanisminpartialseizures.Finally,slow-wavesleepisalso
transmission arising from basal forebrain (Figures 5, 6E, and marked by decreased acetylcholine in the thalamus (Williams
6F)andPPT(Figures4,6A,and6B),respectively. et al., 1994), which also occurred in partial seizures. Activity
To further confirm the validity of our measurements, cholin- changesincortex,thalamus,andbrainstemareprominentdur-
ergictransmissionwas alsoevaluatedusingastimulusknown inganesthesia(Langsjoetal.,2012)andslow-wavesleep(Braun
to produce increased (rather than decreased) physiological et al., 1997) induction. Consistent with a broad suppression
arousal.Attheendoftherecordingsession,theanimalswere of the arousal system, patients with temporal lobe and other
put back into deep anesthesia, and toe pinch was adminis- partialseizuresdemonstrateabimodaldistributionwithrespect
tered to evaluate the cholinergic response. Toe pinch is to impairment of consciousness (Cunningham et al., 2014).
known to increase firing in cholinergic PPT neurons and to Inmostpatients,consciousnessiseitherfullyimpairedorfully
activatecortex (Mena-Segovia etal.,2008).Toe pinchcaused intact, which suggests anoverall decrease in the levelof con-
significant choline increases in both cortex (nine animals; sciousnessratherthanimpairmentofselectedcontentsofcon-
comparing seizure to baseline: mean choline concentration sciousness(Posneretal.,2007).Theseobservationsprovidea
change +0.122 mM ± 0.032 mM; paired t test Holm-Bonferroni humanbehavioralcorrelateforthepresentfindingsofdepressed
corrected, p < 0.05) and thalamus (eight animals; comparing arousalmechanismsduringseizures.
seizuretobaseline:meancholineconcentrationchange+0.040- The arousal system is complex, consisting of at least seven
mM ± 0.014 mM; paired t test Holm-Bonferroni corrected, p < potentialparallelpathwaysofcorticalandsubcorticalexcitation
0.05)(Figure8C). (Saperetal.,2005).Ourexperimentsdemonstrateastrongcor-
relation between cortical depression and a depressed cholin-
DISCUSSION ergic system during partial seizures. The current experiments
donotdeterminewhetherthedecreaseincholinergictransmis-
Theprevailingviewholdsthatdeficitsduringseizuresarecaused sioncausescorticaldepression.However,optogeneticstimula-
bysuddenabnormalorexcessiveactivationofcorticalcircuits. tion of cholinergic PPT neurons decreases cortical slow-wave
Neuron85,561–572,February4,2015ª2015ElsevierInc. 567
A B Figure 8. Choline Signal Decreases in the
CortexandtheThalamusduringPartialSei-
zures
(A) In the cortex, choline recordings decrease
duringpartialseizureswithgradualrecoverydur-
ingthepostictalandrecoveryperiods.
(B) The pattern is the same in the thalamus.
Choline recordings decrease during partial sei-
zures.
(C)Meanictalchangesinthecortexandthethal-
amus relative to 30 s baseline are shownalong
withmeanchangesduringtoepinchtoprovidea
common physiological change for comparison.
Cholinedecreasesinpartialseizureswereallsta-
C tisticallysignificant(pairedttestsHolm-Bonferroni
corrected,p<0.05).Toepinchalsoelicitedsig-
nificantcholineincreasesincortexandthalamus
(paired t tests Holm-Bonferroni corrected, p <
0.05). Error bars in (A)–(C) are SEM. Mean time
coursesin(A)and(B)aredata30spriortoseizure
onset,seizuretimecoursescaledtomeanseizure
duration, and unscaled postictal time course
aligned to seizure end. LO, lateral orbitofrontal
cortex;Thal,intralaminarthalamus.
activity during partial seizures (Furman M et al., ‘‘Optogenetic Low-frequencyoscillationspersistpostictally(Figures4Aand
stimulation of cholinergic mesopontine neurons for preventing 5A,Postictal)astheydoinepilepsypatients(Blumenfeldetal.,
corticaldysfunctionduringseizures’’SocietyforNeuroscience 2004b; Englot et al., 2010), but the mechanism causing a
2013, Poster #53.19/U12), and electrical stimulation of cholin- ‘‘depressed’’ state in the cortex is likely different from the
ergic targets in the intralaminar thalamic CL reduces seizure- mechanismduringtheictalperiod.Wehavehypothesizedthat
associated cortical slowing and promotes behavioral arousal arousalsystemsaresuppressedduringpartialseizuresbecause
(Gummadavellietal.,2015).Althoughcholinergicsystemsplay seizure activity leads to increased inhibition in the subcortical
animportantroleinarousal,itislikelythatsuppressionofother arousal systems. Support for this hypothesis has come from
subcortical arousal systems also participate in loss of con- fornix transection that prevents ictal slow waves and lateral
sciousnessduringseizures.Futurestudiesareneededtodeter- septalstimulationthatcanreproduceslowwaves(Englotetal.,
minetheroleofothercandidatessuchasnoradrenergicneurons 2009). However, postictal firing in the hippocampus, lateral
inthelocus coeruleus,serotonergic neuronsintheraphe,and septum,andanteriorhypothalamusaresuppressedratherthan
orexinneuronsinthelateralhypothalamus. hyperactive (Figure S1), suggesting a different mechanism for
Additionalworkisneededtoexplorethemechanismbehind postictal slow-waves. The BOLD signal in these regions does
thesuppressedneuronalactivityinthesubcorticalarousalsys- remainelevatedintheinitialpostictalperiod(Figure3B,Postic-
temsduringseizures.Itispossiblethatexcitatoryinputtothese tal)despitesuppressedMUAactivity(FigureS1),butthisislikely
systems has been removed or that inhibitory input has been relatedtoaslowerdecayinperfusionrelativetoneuralactivity.
increased. Initial evidence suggests that increased inhibitory Becausewehaveobservedgradualrecoveryofcholinergicfiring
inputstosubcorticalarousalsystemsmayplayarole,including in parallel with decreasing slow waves in the postictal period
thefollowing:(1)lateralseptumandanteriorhypothalamusshow (FiguresS7A–S7C),wecanspeculatethatpersistentdepression
largeBOLDincreases(Figures2and3)duringseizures.These of subcortical arousal may still contribute. However, future
regionsareknowntocontainGABAergicneuronswithinhibitory studies will be needed to fully determine the mechanisms of
projectionstosubcorticalarousalsystems(McGintyandSzymu- depressedarousalandlow-frequencyoscillationsinthepostic-
siak, 2001; Mesulam and Mufson, 1984; Saper et al., 2005; talperiod.
Semba and Fibiger, 1992; Varoqueaux and Poulain, 1999). (2) Impairedconsciousnessinpartialseizureshasamajornega-
Disconnectionofexcitatoryinputstotheseinhibitorystructures tiveimpactonqualityoflifeinpeoplewithepilepsy,causingun-
by cutting the fornix prevents ictal cortical slow activity and safedriving,impairedworkandschoolperformance,andsocial
preventsbehavioralarrest(Englotetal.,2009).(3)Electricalstim- stigmatization(Sperling,2004).Inaddition,impairedarousalin
ulation of the lateral septal nuclei or anterior hypothalamus thepostictalcouldcontributetodifficultyinprotectingtheairway
reproduces cortical slow-wave activity and behavioral arrest in patients found with their face blocked by a pillow causing
(Englot et al., 2009; Sterman and Clemente, 1962). Future suddenunexpecteddeathinepilepsy(SUDEP)(KlosterandEn-
studiesareneededtoinvestigatethepotentialroleofdescend- gelskjøn, 1999). Although stopping seizuresisthe best way to
ing inhibition in decreased subcortical arousal during and preventthesenegativeconsequences,convertingfocalseizures
followingpartialseizures. withimpairedconsciousnesstofocalseizureswithoutimpaired
568 Neuron85,561–572,February4,2015ª2015ElsevierInc.
consciousness would greatly benefit patients with refractory 2008).200acquisitions(150acquisitionsinoneanimal)wereacquiredper
epilepsy. The findings presented here point to new potential 600sexperiment.
therapeuticavenuestoincreasesubcorticalarousalduringand Partialseizureswereinducedapproximately1minfollowingthebeginningof
BOLDacquisitionsasdescribedpreviously(Englotetal.,2008,2009)witha2s
following seizures, including electrical and optogenetic deep
stimulustraindeliveredbetweenthebipolarhippocampalcontactsconsisting
brainstimulationandtargetedpharmacologicalinterventionsto
ofsquarewavebiphasic(1msperphase)pulsesat60Hz.
improvelevelofconsciousness.
Large-scale network switching in the brain is an important fMRIAnalysis
emerging area of neuroscience research. Understanding the Groupt-Map
mechanisms underlying network switching in seizures may Imageanalysiswascompletedusingstatisticalparametricmapping(SPM8,
http://www.fil.ion.ucl.ac.uk/spm/) and in-house software written in Matlab
have implications for understanding normal transitions seen in
(R2009a,MathWorks,Inc.).Baselinewasdefinedbythemeanoftenimages
sleep (Saper et al., 2010) and in reciprocal task-positive and
justpriortoeachseizure.Foreachseizure,meanictalpercentchangeimages
default-mode networks (Fox et al., 2005). We have found werecalculatedusingallimagesfromtheseizurecomparedtobaseline.Ifan
converging evidence based on fMRI mapping, juxtacellular animalhadmorethanoneseizure,ameanictalpercentchangeimagewas
recordings from brainstem neurons, and electrochemical calculatedforthatanimalbytakingthemeanimageacrossseizures.Aone-
transmitter measurements that partial limbic seizures lead to sample t test was then performed on the co-registered percent-change
maps usingSPM8.Afalsediscovery rate(FDR)-corrected pvalueof0.05
depressedsubcorticalcholinergicarousal,whichmayplayacrit-
wasusedforthresholding.
icalroleinlossofconsciousness.Weproposeaparadigmshift
TimeCourses
for studying and treating partial seizures in which loss of con-
Foreachanimal,ROIsweredrawnonitshigh-resolutionanatomicalimageus-
sciousnessisunderstoodasadecreaseinsubcorticalarousal ingarat-brainatlas(PaxinosandWatson,1998).Signalswereobtainedusing
andasuddentransitionintoastateresemblingslow-wavesleep. bilateral ROIsexceptforinthe caseofthe hippocampusandintralaminar
thalamicCLinwhichonlytheleftside(contralateraltotheelectrode)was
usedduetohippocampalelectrodeimagingartifactintherightROI(e.g.,Fig-
EXPERIMENTALPROCEDURES
ures2,APslices(cid:1)3.4mmand(cid:1)4.4mm,and3A).BOLDsignalchangesin
eachROIwerecomputedasapercentchangeoftheROI’sbaselinevalues.
ForfulldetailsofexperimentalproceduresseeSupplementalExperimental
Baselinedataincluded30spriortoseizureonset.Seizuredataincludedthe
Proceduresonline.
entiretimecourseoftheseizurescaledtomeanseizuredurationtoallowfor
averagingacrossanimals.Postictaldatawerealignedtoseizureoffsetwithout
AnimalPreparationandSurgery scaling(Figure3B).Aone-sampletwo-tailedttestwascomputedforeachROI
Allprocedureswereconductedinfullcompliancewithapprovedinstitutional usingthemeanROIpercentchangeduringtheictalacquisitions.Significance
animalcareanduseprotocols.Atotalof138adultfemaleSpragueDawley thresholdwasp<0.05withHolm-Bonferronicorrection.
rats(CharlesRiverLaboratories)weighing202–365gwereusedintheseex-
periments.Allsurgerieswereperformedunderdeepanesthesiawithketamine AcuteLFP,MUA,Juxtacellular,andCholineAnimalPreparation
(90mg/kg)andxylazine(15mg/kg).AftersurgeryanimalsusedinfMRIexper- Abipolarelectrodeforstimulating/recordingLFPwasstereotacticallyplaced
imentswereallowedatleast6daysofrecoverypriortoimaging(seebelow), intheleftdorsalhippocampus.Injuxtacellularrecordings,ahigh-impedance
whereas animals for juxtacellular and neurotransmitter experiments were monopolarelectrodeforrecordinglocalfieldpotential(LFP)andmultiunitac-
switchedtoalow-dose,‘‘light-anesthesia’’ketamine(40mg/kg)andxylazine tivity (MUA) was implanted into the right lateral orbital frontal cortex (LO)
(7 mg/kg) for immediate recordings as described previously (Englot et al., (AP,+4.2;ML,2.2;SI,2.4).Incholinerecordings,toavoidshuntingamperom-
2008). All stereotactic coordinates were taken from Paxinos and Watson etysignalsviatheground,insteadofahigh-impedanceMUA/LFPelectrode
(1998)andweremeasuredinmillimetersrelativetobregma.Followingexper- weusedabipolarLFPelectrodeforrecordingsfromLO.TheLOelectrode
iments,animalsweresacrificedwithEuthasol(Virbac),andbrainswerehar- waspreparedinasimilarfashiontothehippocampalelectrodebutimplanted
vestedforhistologicalanalysisandtoverifyelectrodelocations. inthesagittalplane.
fMRIExperiments ElectrophysiologyRecordingsofMUAandLFP
Ourimplantandimagingmethodshavebeendescribedpreviously(Englot DataAcquisition
etal.,2008).Inbrief,atleast6daysbeforefMRIrecordings,animalswereim- CollectionofextracellularMUAandLFPrecordingsacquiredfrommonopolar
plantedstereotacticallywithanMRI-compatiblebipolartungstenelectrodein electrodeswasdescribedpreviously(Englotetal.,2008,2009;Schriddeetal.,
therightdorsalhippocampus.Torecordscalpelectroencephalogram(EEG) 2008).Briefly,MUAandLFPwereacquiredusingaMicroelectrodeACAmpli-
simultaneouslywithfMRI,apairofcarbonfiberelectrodeswereplacedbilat- fier(model1800;A-MSystems)andbroadbandfilteredfrom0.1Hzto20kHz
erallyoverthecortexbetweenthescalpandtheskullonthedayoftheexper- (3100 gain). Signals were subsequently filtered using a model 3363 filter
iment. After surgery and preparation for fMRI experiments, animals were (Krohn-Hite)intoLFP(0.1Hz–100Hz,315gain)andMUA(400Hz–10kHz,
switched to a low-dose ketamine/xylazine and maintained under this light 320gain).LFPfrombipolarelectrodeswasacquiredusingthesamemodel
anesthesiaregimenduringseizureinductionasdescribedpreviously(Englot 1800amplifierwithabroadbandfilterof1Hz–500Hz(31,000gain).Signals
etal.,2008,2009). weredigitizedandrecordedusingaCEDPower1401andSpike2software
fMRI recordings were acquired on a modified 9.4 T system with Varian (CED).LFPfrommonopolarorbipolarelectrodesweredigitizedat1,000Hz;
spectrometerusingacustom-built2311Hsurfacequadraturecoilfortrans- MUAfrommonopolarelectrodesweredigitizedat20,000Hz.
mittingandreceivingradiofrequencypulses(Hydeetal.,1987).Highspatial MUAandLFPAnalysis
resolution anatomical images were acquired with ten slices in the coronal Recordingswereanalyzedusingin-housesoftwarewritteninMatlab(R2009a,
plane using gradient-echo or spin-echo contrast. For fMRI, a single shot Mathworks,Inc.).Rootmeansquare(RMS)wascalculatedbysquaringthe
spinecho-planarimaging(SE-EPI)sequencewasused:TR,1,000ms;TE, meansignalin1sbinsandthentakingthesquareroot.MUAtimecourses
25 ms; FOV, 25 3 25 mm; matrix, 64 3 64; in-plane resolution, 390 3 werecalculatedusingsuccessive1sbinswith0.5soverlaps.Thetimecourses
390 mm; contiguous slices of 1 mm, with ten slices obtained in the same arereportedasapercentchangefromthe30sofbaselinepriortoseizureiniti-
planesasanatomicalimages.ThefMRIimageswereobtainedwith1sacqui- ation(FigureS1).WeusedRMSoftheMUAsignalasanestimateofactionpo-
sitionfollowedby2sdelaytoenableEEGandLFPsignalstobereadilyinter- tentialfiring,becausethisapproachhasbeenpreviouslyvalidated inboth
pretedduringimaging(Englotetal.,2008;Mishraetal.,2011;Schriddeetal., normal conditions (Shmuel et al., 2006) and during seizures (Englot et al.,
Neuron85,561–572,February4,2015ª2015ElsevierInc. 569