wonoep appraisal: optogenetic tools to suppress seizures and explore the mechanisms of...

WONOEP appraisal: Optogenetic tools to suppress

seizures and explore themechanisms of epileptogenesis*LauraMantoan Ritter, †PeymanGolshani, ‡Koji Takahashi, §¶Suzie Dufour, §Taufik Valiante,

and #Merab Kokaia

Epilepsia, 55(11):1693–1702, 2014doi: 10.1111/epi.12804

Laura Mantoan Ritteris a Neurology Trainee

in London; her

research interests are

the mechanisms of

epileptogenesis and

the development of

novel treatment

approaches in

epilepsy.

SUMMARY

Optogenetics is a novel technology that combines optics and genetics by optical con-

trol ofmicrobial opsins, targeted to living cell membranes. The versatility and the elec-

trophysiologic characteristics of the light-sensitive ion-channels channelrhodopsin-2

(ChR2), halorhodopsin (NpHR), and the light-sensitive proton pump archaerhodopsin-

3 (Arch) make these optogenetic tools potent candidates in controlling neuronal firing

inmodels of epilepsy and in providing insights into the physiology and pathology of neu-

ronal network organization and synchronization. Opsins allow selective activation of

excitatory neurons and inhibitory interneurons, or subclasses of interneurons, to study

their activity patterns in distinct brain-states in vivo and to dissect their role in genera-

tion of synchrony and seizures. The influence of gliotransmission on epileptic network

function is another topic of great interest that can be further explored by using light-

activated Gq protein–coupled opsins for selective activation of astrocytes. The ever-

growing optogenetic toolbox can also be combined with emerging techniques that

have greatly expanded our ability to record specific subtypes of cortical and hippocam-

pal neurons in awake behaving animals such as juxtacellular recording and two-photon

guided whole-cell recording, to identify the specific subtypes of neurons that are

altered in epileptic networks. Finally, optogenetic tools allow rapid and reversible sup-

pression of epileptic electroencephalography (EEG) activity upon photoactivation.

This review outlines the most recent advances achieved with optogenetic techniques

in the field of epilepsy by summarizing the presentations contributed to the 13th ILAE

WONOEPmeeting held in the LaurentianMountains, Quebec, in June 2013.

KEY WORDS: Epilepsy, Interneurons, Gliotransmission, Optic inhibition, Halorho-

dopsin, Channelrhodopsin, Seizure detection.

Opsins are proteins that combine with the vitaminA–derived chromophore retinal (or retinaldehyde) andbelong to a family of photosensory receptors present inall animal kingdoms, where they subserve a wide varietyof functions, from phototaxis in flagellates to eyesight inanimals. The first optical techniques developed to manip-ulate neuronal activity consisted of phototransductioncomponents of Drosophila expressed in neurons1 andcombinations of heterologous expression of ligand-gatedchannels and injected photolabile-caged compounds.2

Substantial progress led to development of two opsinswith the necessary temporal resolution to control neuronsat the resolution of single spikes:3 channelrhodopsin-2(ChR2), a cation channel from Chlamydomonas rein-hardtii mediating neuronal depolarization when activated

Accepted August 17, 2014; Early View publication October 9, 2014.*Department of Clinical and Experimental Epilepsy, Institute of

Neurology, University College London, United Kingdom; †Department ofNeurology, David Geffen School of Medicine at University of California atLos Angeles, Los Angeles, California, U.S.A.; ‡Department of Neurologyand Neurological Sciences, Stanford University School of Medicine,Stanford, California, U.S.A.; §Division of Fundamental Neurobiology,Toronto Western Research Institute, Toronto, Ontario, Canada; ¶Instituteof Biomaterials and Biomedical Engineering, University of Toronto,Ontario, Canada; and #Experimental Epilepsy Group, Epilepsy Center,Lund University Hospital, Lund, Sweden

Address correspondence to Laura Mantoan Ritter, Department of Clini-cal and Experimental Epilepsy, Institute of Neurology, University CollegeLondon, Queen Square, WC1N 3BG London, U.K. E-mail: [email protected]

Wiley Periodicals, Inc.© 2014 International League Against Epilepsy

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SPECIAL REPORT

Table

1.Detailsofopsintypes,targetingtechnique,andexpressionsystems

Opsintypes

Abbreviation

Peak

activationwavelength

Comments

Excitatory

opsins

Cationchannels:allowpositive

charge

into

theneuronon

illumination,depolarizingthecellmembrane

Channelrhodopsin-2

ChR2

~470nm

Fromthealga

Chlam

ydom

onas

reinhardtii3

ImprovedvariantsofC

hR24

(1)Selectivemutagenesis,e.g.,

ChR2(H

134R)11(2)

Chimerae,e.g.,ChIEF1

2

(1)ChR2(H

134R):470nm

(2)ChIEF:450nm

Achievedbyeither(1)selectivelymutagenizingChR2to

substitute

aminoacidsinvolvedinChR2channelkinetics,itsspectralor

membranetraffickingproperties,or(2)bycreatingchimeraeof

ChR2andotheropsins

Channelrhodopsinsfromother

organisms

VChR16

545nm

FromVolvoxcarteri.

VChR1has

theadvantage

tobemore

red-shiftedthan

ChR2,

implyingthatboth

channelscanbeusedinthesamepreparationto

targetandexcite

differentcellpopulations

ImprovedvariantsofV

ChR1

C1V113

540nm

Afar-redshiftedversionofV

ChR1,w

hichiscompatiblewithFura-2

andGCaM

PCa2

+imagingtechniques

Bi-stable

opsins7

e.g.,ChR2-stepfunction

opsins

Activation:470nm

Inactivation:590nm

ChR2variantswhose

photocurrentscanbeinactivatedon

illuminationwithawavelengthdifferentfromtheactivation

wavelength,therebyallowingsteplikecontrolofthemembrane

potential

Inhibitory

opsins

Pump-negative

charge

into

theneuronorallowpositive

charge

outof

theneurononillumination,hyperpolarizingthecellmembrane

Chloridepumps

Halorhodopsin14,15

e.g.,eNpHR3.0

590nm

FromthearcheaNatronomonas

pharaonis

Chloride-conducting

channelrhodopsin9

ChloC

476nm

Channelrhodopsinconvertedinto

alight-gatedchloridechannel

Protonpumps

Archaerhodopsin-3

10

Arch/ArchT

566nm

FromHalorubrumsodomense

Leptosphaeriamaculansopsin10

Mac

565nm

FromthefungusLeptosphaeriamaculans

Biochemicalmodulators

8opto-X

Rs

500nm

Chimericopsin–G

protein–coupledreceptorprotein(G

PCR);allow

opticalcontrolofG

PCRsignallingcascades

Cell-typespecifictargetingtechniques

Transcriptionaltargeting

Targetingopsinsattheleveloftransgeneexpression

Promoters

ApromoterconsistsofD

NAelementsflankingthetranscription

initiationsite

ofa

gene,w

hichareinvolvedinrecruitingthe

transcriptionmachinery

forthesuccessfultranscriptionofa

protein-

codinggene

Examples

Targets

Synapsin-1,elongationfactor

1a(EF-1a)

14

Strongpan-neuronalpromoters

Calciumcalmodulin-binding

kinase2a(C

amk2a)

16

Cam

k2aisspecificforexcitatory

neurons

Glutamicaciddecarboxylase

(GAD),parvalbumin(PV)

Specificforinhibitory

neurons

Glialfibrillary

acidicprotein

(GFA

P)

Specificforastrocytes

Continued


1694

L. Mantoan Ritter et al.

Transductionaltargeting

Ifusingviruses,targetingispossibleatthelevelofvectorentryinto

thecell

Viralenvelopes

Achievedbypackagingthe

opsincarryingviralvector

usingenvelopeglycoproteins

ofdifferenttypesof

neurotropicvirusessuch

as

Rabies-,M

okola-,Ross-River-

,HSV

-,andVSV

g-viruses

Viralserotypes

Usingrecombinant

adeno-associatedvirus

serotypeswithtropismfor

neuronsorglia

Opsinexpressionsystem

s4Exam

ples

Advantages

Disadvantages

Viralvectors

17

Lentivirus(LV),adeno-associatedviralvector

(AAV),Herpes-simplexvirus(H

SV)

Ableto

transduce

term

inallydifferentiatedcells,to

penetratetheblood–b

rainbarrier(forAAVs),to

eludetheimmunesystemandhavetherapeutic

potential

Manypromotershavenotyetbeencloned,hence

viruses

cannotbeusedto

targetthese

celltypes(e.g.,PV-

interneurons).Potentiallyimmunogenic(forAAVs)and

oncogenic(forintegratingLVs)

Transgenicanimals

e.g.,vG

AT::C

hR2-eYFP

mice(expressing

ChR2undertheinterneuron-specificmouse

vesicularGABAtransporter(vGAT)

promoter)

Itispossibleto

clonean

opsingenedownstream

ofa

specificpromoterevenifthepromoterhas

notbeen

fully

characterized.W

idespread

expression

throughoutthenervoussystem.Expressioncanbe

studiedthrough

development

Cost,timeanddifficultyto

generate

transgenicanimals

Cre-Loxsystems

GAD2-C

reorPV-C

remiceanddouble-

floxedChR2-m

Cherryviralvector

(rAAV-EF1a-DIO

-ChR2(H

134R)-mCherry:

transgenicmiceexpresstheenzymeCre-

recombinase(C

re)undereithertheGADor

thePV-promoter.Cre

cutstw

ospecific

mirrorDNAsequences(“floxed”).T

hegene

betw

eenthese

sequencesistheninverted

andreinsertedinto

theDNA.T

heviral

vectorinjectedinto

GAD-orPV-C

remice

containsan

invertedChR2genebetw

eenthe

2floxedsequences.W

hilsttheviralvector

may

enteranytypeofcell,onlyGADorPV

interneuronsexpressCre,w

hichallowsthe

invertedChR2geneto

beexcised,

reinsertedcorrectlyandthenexpressed

Allowstargetedexpressioninneuronswhose

promoterscannotbeclonedinto

avirus.ManyCre-

loxtransgenicanimalsandvectorsarereadily

availablefromcommercialsources

Cost,time,anddifficultyto

generate

newtransgenicanimals

andclonenewvectors

Table

I.Continued.


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Optogenetic Tools to Study Epilepsy

by 470 nm blue light, and the inhibitory halorhodopsin(NpHR), a chloride pump found in Natronomonas phara-onis and activated by 590 nm yellow light (Fig. 1A).Since, further engineering of optogenetic tools hasyielded channelrhodopsin variants with different absorp-tion spectra and kinetic properties,4,5 excitatory opsinsfrom other species, for example, Volvox carteri channel-rhodopsin,6 step-function opsins,7 chimeric opsin G pro-tein–coupled receptor (GPCR) proteins to allow opticalcontrol of GPCR signaling,8 and new inhibitors such asa chloride-conducting channelrhodopsin variant (ChloC),9

archaerhodopsin-3 (Arch) and an opsin from the fungusLeptosphaeria maculans (Mac), two proton-pumps acti-vated by yellow and blue light, respectively10 (Table 1).By allowing cell-type specific and precise modulation ofsingle neurons or whole populations of neurons17 (Fig.1B-D) this fascinating technique has revolutionized neu-roscience.17 (Fig. 1B–D). Expression of these microbiallight-sensitive proteins has been used to study specificclasses of neurons in vitro3,14 and in intact brain tissuein vivo in vertebrate16,18 and invertebrate models.19 Tar-geting specific neuronal subpopulations can be achievedusing cell-type specific promoters in viral vec-tors14,16,19,20,21 and in transgenic animals22 or Cre-Loxsystems, or by employing both23 (Table 1). To allowoptical stimulation in vivo, integrated fiberoptic and op-togenetic technologies were developed and currently con-sist of direct implantation of a custom-made fiberopticcannula into an area of viral injection (Fig. 1E) or intransgenic mice expressing opsins under a specific pro-moter.24–26 In mammals, the interface has been imple-mented in rat, mouse, and monkey models, and was usedto target specific neurons without evidence of a func-tional immune response in vivo.16,18,27,28 More recently,further development of viral vectors and opsins has madeit possible to selectively stimulate projection tracts ratherthan only somata,29 and to achieve pathway-specific tar-geting of specific subpopulations of neurons.15 Giventheir versatility and electrophysiologic characteristics, op-sins constitute a reliable toolbox enabling systematicanalysis of epileptic neural circuits, and are being usedto provide insights into the physiology and pathology ofneuronal network organization and synchronization.

More recent applications have focused on opsins aspotential therapeutic tools,30–32 and photoactivation ofprokaryotic light-sensitive ion channels or transportersexpressed in neurons has been explored in vitro and invivo to suppress seizure activity “on demand”23,33,34

(Fig. 1E).This review will summarize the presentations on

advanced optogenetic techniques used to explore epilepticnetworks and to interrupt seizures contributed at the 13thILAE WONOEP (International League Against EpilepsyWorkshop on Neurobiology of Epilepsy) meeting held inthe Laurentian Mountains, Quebec, in June 2013.

Combined Optogenetic

Techniques for Recording

Network Dynamics in Animal

Models

Epileptogenesis reorganizes cortical and hippocampalcircuits, but the effects of these changes on the emergentnetwork properties of precisely identified interneuronsand pyramidal neurons are poorly understood.35 This levelof understanding is essential for determining the circuitcomponents that are critical for seizure initiation. Theexpanding array of optogenetic tools and vectors used totarget and stimulate specific subclasses of neurons4,5 cannow be combined with the emerging techniques that havegreatly expanded our ability to record specific subtypes ofcortical and hippocampal neurons in awake behavinganimals. These emerging techniques include juxtacellularrecording,36 two-photon guided whole-cell recording,37,38

imaging of genetically encoded calcium indicators (GE-CIs),39 and silicon nanoprobe recording40,41(Fig. 2A–F).Juxtacellular recordings can be used to record actionpotentials from single neurons (Fig. 2A,B), which cansubsequently be filled with biocytin and anatomically andimmunocytochemically identified post hoc. Although thistechnique allows recordings from single neurons only ineach brain region, its advantages include the following:the recordings (1) do not alter intracellular contents; (2)allow precise identification of the recorded neuron, and(3) yield recordings with perfect single-neuron isolation.36

Two-photon microscopy allows identification of specificcell types expressing fluorophores using Cre-Lox strate-gies:42 it can be effectively combined with in vivo whole-cell recordings to record the subthreshold membranepotential dynamics of specific cortical interneuron typesduring rest and locomotion.43 Two-photon calcium imag-ing (Fig. 2E,F) can be used to record the activity patternsof large ensembles of neurons in awake animals, with nosampling bias for cells with higher firing rates,44,45

although calcium indicators and scanning techniques, stilllimit the temporal resolution of the recordings.44,45 Newgenetically encoded calcium indicators show exceptionalpromise, because they reliably report single action poten-tials and make it possible to image the same cells overseveral weeks,46 potentially allowing recordings of thesame cells before and after epileptogenesis. In addition,they allow the precise location of each cell and its spatialrelationship to other neurons to be ascertained. Further-more, with these indicators it is also possible to recordactivity patterns from long-range projecting axons ofgenetically identified neurons, making it possible to imagethe different inputs to the same population of neurons.Sparse labeling strategies have made it possible to imagethe activity patterns of individual dendritic spines, allow-ing one to determine how incoming sensory and internally


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A

C

E

D

B

Figure 1.

Schematic of optogenetic tools and their modes of action. (A) Opsin types. Diagram showing the three major classes of opsins used to

date (from left to right): (Left) Excitatory opsins are light-activated cation channels (represented here by channelrhodopsin-2 [ChR2]),

which allow positive charge into the neuron on illumination, and hence depolarize the cell membrane. The peak activation wavelength for

ChR2 is ~470 nm, corresponding to blue light. (Center) Inhibitory opsins (represented by the chloride pump halorhodopsin [NpHR])

hyperpolarize the neuronal membrane by either pumping negative charge into the cell (NpHR) or extruding positively charged protons

(as in the case of archaerhodopsin (ArchT) on illumination. NpHR is maximally activated by 590 nm yellow/green light. (Right) Chimeric

opsins (OptoXR) are fused with adrenergic G protein–coupled receptors and can act as biochemical modulators by light-induced increase

of cAMP or G protein signaling. (B) Opsin gene expression and identification. Targeted expression of opsins in neurons can be achieved

by using cell-type specific promoters in transgenic animals or by using viral vectors. The opsin gene is tagged with a fluorescent protein

gene to allow identification of the opsin-expressing cells. Depicted, a confocal fluorescence micrograph showing expression of the adeno-

associated virus AAV-eNPAC (in green), carrying both ChR2 and NpHR tagged with green-fluorescent protein (GFP) in hippocampal

pyramidal cells. Overlay with DAPI (40,6-diamidino-2-phenylindole) to identify cell nuclei. (C) ChR2 activation in vitro. Sample trace of a

patch-clamped CA3 pyramidal neuron expressing AAV5-eNPAC. Illumination with 2 s pulses of 473 nm laser light (two pulses, 1 Hz)

drives sustained action potential firing. (D) NpHR inhibits action potentials in hippocampal pyramidal cells. A CA1 neuron expressing

AAV5-eNPAC is stimulated with a 593 nm laser. Yellow light hyperpolarizes the membrane and inhibits action potential firing during

current injection (30 pA, 20 msec pulses). Membrane potential (Em). (C,D) Reproduced with permission fromMantoan and Kullmann.61

(E) Optogenetic inhibition in vivo. Schematic of the implanted headstage (magnified right inset: optic cannula and electrode implanted

above the NpHR-expressing neurons in green) for simultaneous EEG recording and optical stimulation. (Left) representative EEG traces

before and during 561 nm laser illumination, in an epileptic animal, showing a decrease in epileptiform activity on illumination of NpHR.

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generated activity patterns are integrated at the level ofsingle dendrites.46

Finally, miniaturized fluorescence microscopes can beimplanted onto mice to perform imaging of hippocampalactivity patterns in the same cells over months, in unre-strained animals that are free to explore their environment.47

For recordings in deep structures, or during instances whenprecise temporal resolution is critical, advances in the gen-eration of silicon nanoprobes now allow recordings fromlarge numbers of single units in deep structures40,41 simulta-neously with local field potentials, and can readily be scaledfor recordings from hundreds of cells in multiple brainregions. Extracellular electrophysiologic recordings can becombined with optogenetics to identify specific cell types.However, all these strategies bare some limitations in thatfor many cell types in the brain, no specific Cre line or viralstrategy exists to express ChR2 selectively. In addition, it isstill difficult to maintain recordings from the same neuronsfor more than a few days.

Many of these experiments are performed in head-fixedanimals that are free to run or rest on spherical36–38,48 ortraditional treadmills.49 To identify the specific subtypes ofneurons that are altered in epileptic animals and to establishtheir activity patterns in distinct brain states, Golshani et al.,have developed techniques for performing stable whole-cellor juxtacellular electrophysiologic recordings and two-pho-ton calcium imaging from cortical and hippocampal neu-rons in head-fixed epileptic mice. Recordings wereexceptionally stable, allowing high quality recordingsfrom identified neurons for up to 90 min in running mice.These techniques were combined with a virtual reality

environment spanning 270 degrees of mouse vision, wheremovements of the mice on the treadmill resulted in move-ment of a virtual T-maze. During these tasks, neurons canbe addressed optogenetically by stimulating channelrho-dopsin expressed in different cell types.49

Interrogating the Role of

Interneurons in Seizure Models

Much evidence suggests that altered interneuron activityplays a significant role in the synchronization of networksand the development of the hyperexcitability observed inseizures.50,51 Conversely, enhancement of c-aminobutyricacid (GABA)ergic function has undoubtedly an anticonvul-sant effect, as evidenced by the mechanism of action andefficacy of many antiepileptic drugs (AEDs). Selective acti-vation of inhibitory interneuron populations could thereforebe explored as an alternative strategy for suppressing epi-leptiform activity and decreasing network excitability.However, previous studies demonstrated abnormalities ofGABAergic function in several genetic and experimentalepilepsy models,50,52 and little is known about the exactmechanisms through which inhibitory GABAergic neuronsshape epileptiform activity.51 Advances in optogeneticsnow offer the opportunity to address such questions byselectively activating specific subclasses of interneurons.Kokaia’s group.53 utilized glutamic-acid decarboxylase2-Cre recombinase (Gad2-Cre), parvalbumin-Cre-recombin-ase (PV-Cre), and somatostatin-Cre-recombinase (SST-Cre)mouse lines and injected a double-floxed ChR2-mCherry

A C E

B D F

Figure 2.

Combined optogenetic techniques. (A) Illustration demonstrating configuration of juxtacellular and local field potential (LFP) electrodes

during juxtacellular recordings from a CA1 hippocampal parvalbumin-positive basket cell in a head-fixed mouse free to rest or run on a

treadmill. The camera-lucida drawing shows dendrites in red and axons in blue, and is adapted from Varga et al.36 (B) Local field potential

and juxtacellular recording from a parvalbumin-positive basket cell during locomotion-associated theta oscillations. (C) Two-photon

guided whole-cell recording from a parvalbumin-positive visual cortical neuron (yellow) in a PV-Cre X Ai9 mouse, where parvalbumin

positive neurons are labeled with Td-Tomato (red); adapted from Polack et al.43 (D) In vivo whole-cell recording from a visual cortical

neuron in an awake head-fixed mouse free to rest or run on a spherical treadmill (unpublished data, Michael Einstein and Pierre-Olivier

Polack). (E) Two-photon calcium imaging from a population of layer 2/3 motor cortical neurons transfected with GCAMP6 (unpublished

data, Tim Indersmitten). (F) Calcium traces from a subset of the neurons depicted in (E), demonstrating spontaneous activity in motor

cortex during periods of rest in an awake mouse. Scale bars in (A), (C), and (E): 50 lm.

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viral vector (rAAV-EF1a-DIO-ChR2(H134R)-mCherry,containing an inverted version of the ChR2 gene) into theirhippocampi, resulting in expression of ChR2 in hippocam-pal interneurons as a whole or in parvalbumin (PV)- as wellas SST-positive neurons, respectively. Global optogeneticactivation of mixed interneuron populations by activatingGad2-driven ChR2 with blue light in hippocampal slices,effectively suppressed epileptiform activity induced by 4-aminopyridine (4-AP) and zero Mg2+. This attenuation ofbursting epileptiform activity was shown to be caused byinhibition of principal cells by GABA released from inter-neurons. Optical stimulation of PV- or SST-interneuronsalone was less effective in inhibiting epileptiform events.Moreover, postsynaptic currents in pyramidal neurons werelarger and with slower decay times if generated by GABAreleased from interneurons in Gad2-Cre mice as comparedto PV-Cre mice. These data are in keeping with the resultsof recent studies demonstrating that closed-loop optogeneticactivation of PV-interneurons attenuates epileptic seizuresin the hippocampus of freely moving mice,23 supporting thenotion that interneuron-based optogenetic approaches couldbe an effective strategy for suppressing seizures. To addressthe role of interneuron activation during seizures and theirdependence on brain states, Dufour and Valiante developeda multimodal recording system that incorporates wide-fieldin vivo imaging, electrophysiologic recording, and opticalactuators. They used vGAT::ChR2-eYFP mice, expressingChR2 under the interneuron-specific mouse vesicularGABA transporter (vGAT) promoter. Local microinjectionsof 4-aminopyridine were performed in the somatosensorycortex of anesthetized mice to induce seizures. Light stimuli(473 nm) were delivered through an optical fiber placedabove the 4-AP–treated cortical surface. Simultaneous localfield potential recording and intrinsic signal optical imaging(Fig. 3) showed that interneuronal activation with a shorttrain of light stimuli had a transient attenuating effect duringictal activity (Fig. 4 top trace). Conversely, activation of in-terneurons during the interictal phase could lead to seizures(Fig. 4, bottom trace). Taken together, these results suggestthat specific activation of interneurons is sufficient toinduce a change in the epileptic state, and that their effect onthe network may be dependent on the underlying brainactivity state and parameters of optogenetic stimulation inthe live animal.

Optogenetic Control of

Gliotransmission

Astrocytes are crucially involved in the modulation ofneuronal excitability by removing and recycling extracellu-lar neurotransmitters such as glutamate and maintaining lowextracellular concentrations of potassium, a key regulator ofneuronal excitability. In addition, resting and reactiveastroglia contribute to the regulation of blood–brain barrier

function and the uptake of albumin, which has beenimplicated in the process of epileptogenesis.54,55 Finally,much experimental evidence supports the existence of activ-ity-dependent release of neurotransmitters from astrocytesthat signal back to nearby neurons resulting in gliotransmis-sion.56–58 Increasing evidence suggests that astrocytesexpress G protein–coupled receptors (GPCRs) that are acti-vated by synaptic activity. In turn, GPCR activation ishypothesized to increase intracellular Ca2+ through canoni-cal IP3-dependent pathways that result in feedback signalingfrom astrocytes and thereby gliotransmission. The conceptof gliotransmission has been controversial, due to the inabil-ity to activate astrocytic GPCRs and subsequent Ca2+ signal-ling with specificity. To circumvent this barrier, Takahashiet al. expressed opto-a1AR, a rhodopsin-adrenergic receptorchimaera, specifically in astrocytes of the cortex and hippo-campus. This novel opsin replaces the intracellular loops ofGt-coupled vertebrate rhodopsin with those from Gq-cou-pled a1-adrenergic receptor, to enable the control of intra-cellular signaling with light.8 Specificity for astrocytes wasachieved by expressing the opsin construct under the astro-cyte-specific minimal promoter gfaABC1D21 and by target-ing astrocytes with adeno-associated virus serotype 2/5,known to have glial tropism. The vector GFAP::opto-a1AR-eYFP expressed in hippocampal astrocytes as early as2 weeks postinjection, and with use of epifluorescent illumi-nation or focal laser scanning, light-dependent responseswere observed in nearby pyramidal neurons. Intriguingly,the response in hippocampal CA1 pyramidal neurons uponstratum radiatum astrocyte activation was heterogeneousand dependent on the duration of light: A short light pulse(500–750 msec) led to depolarization and spiking in neigh-boring neurons, whereas a longer light pulse (>3 s) led todepolarization and spiking followed by a prolonged potas-sium channel-dependent hyperpolarization that returned tobaseline only after several minutes. These responses werethought to be caused by the release of gliotransmitters trig-gered by optogenetic activation of Gq-mediated signaling inastrocytes, as the neuronal depolarization and hyperpolariza-tion responses (but not the spiking activity) were also pre-served when presynaptic transmission was blocked withtetrodotoxin, suggesting that these effects are independentof action potentials. Further work will determine the glio-transmitters released by opto-a1AR activation and investigateastrocytic involvement in epileptogenic circuits induced bypilocarpine status epilepticus.

Optogenetics as Therapeutic

Tools: Detecting and Silencing

Seizures in Chronic Epilepsy

Models

The main therapeutic goal in any form of epilepsy is free-dom of seizures with a well-tolerated treatment. But curing


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epilepsy also entails striving to find a more specific way oftargeting treatments to maximize benefits and avoid sideeffects.59 Optogenetic tools are ideally suited to accomplishthis task by allowing cell-type specific targeting and trans-duction of eloquent cortex, combined with seizure suppres-sion “on demand,” thereby minimizing the risk ofinterfering with normal brain function. Kokaia et al. werefirst to demonstrate that this approach was feasible, as selec-tive hyperpolarization of principal neurons by activation ofNpHR in the hippocampus inhibited stimulation-inducedepileptiform activity in organotypic hippocampal slice cul-tures.60 Three studies have shown that optogenetic silencingof epileptiform activity is successful in chronic models of

focal epilepsy studied in vivo: Mantoan et al. used injectionof tetanus toxin into rodent primary motor cortex to modelhigh frequency activity bursts found in the seizure-onsetzones of patients with focal epilepsy.34 Animals wereimplanted with a wireless transmitter that allowed continu-ous EEG telemetry and off-line analysis. This wasintegrated with an automated event classifier that used asupervised learning algorithm to compare with EEG exemp-lars classified as seizure-related, artifacts or occurring withnormal behaviors (http://www.opensourceinstruments.com/Electronics/A3018/Seizure_Detection.html). Co-injectionof high-titer lentivirus-carrying NpHR under the Camk2apromoter allowed rapid and reversible suppression ofepileptic EEG activity upon laser photoactivation ofNpHR.34 Photoactivation of the chloride pump halorhodop-sin in the rat thalamus via a closed-loop system was alsoused to attenuate poststroke seizures in rodent sensorycortex.33 Finally, both optical inhibition of principal cellsand light-mediated activation of PV-interneurons wassuccessful in arresting seizures in a kainate model of tempo-ral lobe epilepsy.23 Optical modulation of neurons in theepileptogenic zone, combined with wireless telemetry andseizure detection algorithms, represents a promising newplatform to develop an automated device to stop seizuresacutely, akin to an implantable defibrillator.

Conclusions

Optogenetics is a relatively new approach to selectivelyaddress specific neuronal populations and activate or sup-press their action potentials with millisecond time resolu-tion. Thus optogenetic tools provide the yet-unparalleledopportunity to study detailed neuronal mechanisms behind

Figure 3.

Intrinsic signal optical imaging to visualize seizures. Example showing a time series of intrinsic signal optical images (imaging wavelength:

680 nm) obtained from the somatosensory cortex of a mouse during a seizure triggered by an optogenetic interneuronal activation (at

t = 0 s). The seizure appears as a dip in the intrinsic signal (in yellow/red). Images are shown as relative intensity (I) fluctuations (DI/I).Scale bar represents 200 lm.

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Figure 4.

Optogenetic interneuron activation and seizures. A 10 Hz blue

light stimulus (pulse duration = 40 msec, train duration = 1 s) has

different effects on cortical local field potentials (LFP) depending

on the baseline EEG of a VGAT::CHR2-eYFP mouse (The Jackson

Laboratory, Jackson, ME, U.S.A.): Case 1 shows how cortical LFP

activity is attenuated if interneurons are stimulated during a seizure

(induced by 4-AP). Case 2, shows that the same blue light stimulus

delivered during an interictal phase leads to epileptiform rhythmi-

cal LFP activity.

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specific brain states and functions. Because alterations innetwork excitability and connectivity are the major hall-mark of the pathophysiologic processes associated with epi-lepsy, optogenetic techniques are particularly suited toexplore mechanisms of epileptogenesis and ictogenesis.Studying excitatory and inhibitory neuronal populationsseparately, is advantageous in order to understand theirrespective contribution to ictal and interictal epileptiformactivity. The data presented show that opsins are suitabletools to modulate and study excitatory and inhibitory net-works in epileptic tissue and that optogenetics combinedwith in vivo whole-cell recordings and imaging techniqueswill undoubtedly expand our understanding of circuit dys-function in chronic epilepsy by allowing unprecedentedinsights into epileptogenesis.

We have outlined the most recent advances achieved withoptogenetic techniques in the field of epilepsy, and wewould like to conclude by focusing on the many researchquestions that await investigation with these tools. Opsinsare ideally suited to dissect the contribution of individualpopulations of interneurons on network excitability and willhelp us delineate the role of selective activation of specificinterneuron populations in regulating synchrony, seizureactivity, and brain states. It will be crucial to explore effectsand outcomes of all specific components of the optogeneticapplications, such as cell-specific expression, opsin activa-tion kinetics and parameters, activity-dependent state of thebrain circuits, and their specific response to optogeneticstimulation, light intensity, as well as its paradigms (e.g.,ramp- vs. pulse-light stimulation). Optogenetic tools will bepivotal in addressing the properties of gliotransmission incortex or hippocampus, its effects on excitatory and inhibi-tory cells, and its contribution to epileptogenesis. Futureexperiments should also address the heterogeneity of glio-transmission with respect to different circuits and in devel-opment. Finally, optogenetic treatment approaches forepilepsy are feasible and may in the future become viablefor human patients. Some challenges, however, remainbefore considering opsins as future therapeutic tools: Betterand faster inhibitors are required to generate large photocur-rents, a vector capable of targeted insertions into the hostgenome and a better understanding of inducible and cell-subtype specific promoters will reduce the current risk ofmutagenesis, oncogenesis, and the lack of control overtransgene expression posed by current vectors. Safety stud-ies should also address whether long-term expression of op-sins could trigger an immune response. We envisage thatthe development of reliable seizure detection algorithmsvalidated in human epilepsy and of miniaturized biocompat-ible devices, will eventually make it possible to manufac-ture an implantable closed-loop optogenetic system capableof triggering the generation of a “defibrillator” light pulseonce the electrical signature of a seizure onset is detected,thus yielding a radically new treatment alternative forhuman disease.

Acknowledgements

This work was supported by the Swedish Research Council, NS12151,NS39579, N007280, 1F32NS077623 (D.K.T.) and the Canadian Institutefor Health Research (S.D. and T.V.). S.D. received a fellowship from theMITACS elevate program and the Savoy Foundation. L.M.R. was sup-ported by grants from the Brain Research Trust and the Guarantors of Brain.P. G. was supported from the VA Merit Award (BX001524 01A1), theWhitehall Foundation Award (2012-05-83), and the National Institutes ofHealth (NIH) RO1 Award (1RO1MH101198-01). We would like toacknowledge Joshua A. Dian, Michael Chang, Milad Javaherian, ChristinaKaba, and Tristan Shuman for their work; Dimitri Kullmann, Mary Collins,Sylvain Williams, and Antoine Adamantidis for the helpful discussionregarding these projects; and Christopher Ryan from Q-imaging. We wouldlike to thank Dr Michael Zandi and Rachael Hansford of Advances in Clini-cal Neuroscience & Rehabilitation for granting permission to reproduceparts of a previously published figure.

Disclosure

None of the authors has any conflict of interest to disclose. We confirmthat we have read the Journal’s position on issues involved in ethical publi-cation and affirm that this report is consistent with those guidelines.

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