Microcircuits and their interactions in epilepsy: is the focus out of focus?

Transcription

1 F O C U S O N E P I L E P S Y review Microcircuits nd their interctions in epilepsy: is the focus out of focus? Jenne T Pz 1 & John R Huguenrd 2 Epileptic seizures represent dysfunctionl neurl networks dominted y excessive nd/or hypersynchronous ctivity. Recent progress in the field hs outlined two concepts regrding mechnisms of seizure genertion, or ictogenesis. First, ll seizures, even those ssocited with wht hve historiclly een thought of s primry generlized epilepsies, pper to originte in locl microcircuits nd then propgte from tht initil ictogenic zone. Second, seizures propgte through cererl networks nd engge microcircuits in distl nodes, process tht cn e wekened or even interrupted y suppressing ctivity in such nodes. We descrie vrious microcircuit motifs, with specil emphsis on one tht hs een rodly implicted in severl epilepsies: feed-forwrd inhiition. Furthermore, we discuss how, in the dynmic network in which seizures propgte, focusing on circuit choke points remote from the initition site might e s importnt s tht of the initil dysfunction, the seizure focus. Epilepsy reserch nd neuroscience owe much to insights gined from operting on the humn rin. In the first hlf of the lst century, neurosurgeon Wilder Penfield nd his collegue Herert Jsper pioneered incredile dvnces, such s chrcterizing motor nd sensory mps nd descriing the form of cererl electricl ctivity during seizures 1. Their findings hve inspired decdes-long inquiry imed t understnding nd treting epilepsy. Since then, we hve found mny chnges in structure nd/or function in the epileptic rin of humns nd nimls, such s ltered morphology nd excitility of individul neurons, chnges in expression of neurotrnsmitter receptors, strocytic nd lood-rin-rrier dysfunction, neuroinflmmtion, nd gins or losses of individul circuit components, which would render neurl network hyperexcitle. These studies hve documented moleculr nd/ or ntomicl chnges ssocited with the epileptic rin nd hve een comprehensively descried elsewhere (for exmple, see ref. 2). Despite these insightful studies, there is still no cure for epilepsy. Existing tretments only im to control seizures nd hve sustntil side effects, nd more thn one third of ll epilepsies remin uncontrolled. More recently, technologicl dvnces hve egun to provide detiled descriptions of microcircuit function in oth humns nd niml models of epilepsy. The results of these stte-of-the-rt pproches such s pired (or even higher order) intrcellulr recordings, high-density multi-site extrcellulr rrys, ctivity-dependent reporter dyes nd proteins, nd optogenetics re eginning to provide unique insight into how networks t the micro-scle orgnize nd contriute to generting, propgting nd modulting seizure ctivity. These findings 1 Gldstone Institutes nd University of Cliforni, Sn Frncisco, Cliforni, USA. 2 Deprtment of Neurology nd Neurologicl Sciences, Stnford University School of Medicine, Stnford, Cliforni, USA. Correspondence should e ddressed to J.H. (john.huguenrd@stnford.edu) nd J.T.P. (jenne.pz@gldsone.ucsf.edu). Received 3 Septemer 2014; ccepted 16 Jnury 2015; pulished online 24 Ferury 2015; doi: /nn.3950 chllenge the estlished, yet somewht simplistic, view tht epilepsy simply results from imlnces etween excittion nd inhiition. These dvnces re strting to revel criticl circuit junctures or choke points, potentilly outside of the ictogenic network, tht likely represent trgets for highly specific nd effective nti-epileptic therpies. In this review, we discuss epileptic choke points in the context of severl microcircuit motifs implicted in niml models of epilepsy, s well s those tht hve een confirmed in humns. We will consider the following microcircuit motifs (Fig. 1): 1) feedforwrd inhiition, in which excittory inputs from extrinsic rin regions recruit locl inhiitory networks tht tune the strength nd form of the efferent signl; 2) feed-ck inhiition, in which loclly ctivted inhiitory neurons shpe recurrent excittory ctivity; 3) counter-inhiition, in which locl connections etween inhiitory neurons tht, when ctive, cn decrese output of inhiitory cells nd induce disinhiition or lter oscilltory coupling; nd 4) locl recurrent excittory circuits, common motif in corticl networks in which ~80% of neurons nd synpses re excittory. We lso riefly consider relevnt circuits outside of the microcircuit. These considertions include longer-rnge excittory, inhiitory nd neuromodultory connections tht link nd influence locl microcircuit ctivities. For ech of these motifs, we will identify dysfunctions tht hve een descried t the microcircuit level, illustrte the relevnce of these defects to epileptic seizures nd highlight potentil therpeutic pproches tht might profitly improve tretment of persons with epilepsy. Notly, these motifs do not exist in isoltion, ut re emedded in lrger networks; the fine lnce etween these motifs dicttes the dynmics of lrgescle networks. We focus on the concept tht epileptic seizures emerge from dysfunction of specific microcircuits, which then progressively engge other microcircuits to ctivte the full seizure network n overll process known s ictogenesis. In this context, ictogenic choke points re ny microcircuits or ridges etween microcircuits tht re required for full expression of seizures. nture neuroscience volume 18 numer 3 march

2 Feed-forwrd inhiition Feed-ck inhiition Counter inhiition Recurrent excittion Figure 1 Microcircuit motifs whose dysfunctions hve een identified in epilepsy. Feed-forwrd inhiition: excittory inputs from remote rin regions recruit locl inhiitory networks tht control the strength of the efferent signl. Feed-ck inhiition: locl ctivtion of inhiitory neurons controls locl recurrent excittory ctivity. Counter-inhiition: locl connections etween inhiitory neurons shpe network-inhiitory output. Recurrent excittion: mjor mode of connectivity in corticl networks. Purple nd red represent excittory glutmtergic nd inhiitory GABAergic neurons, respectively. Feed-forwrd inhiition In the lst decde, epilepsy reserch hs provided compelling results regrding the prticulr importnce of feed-forwrd inhiition (Fig. 2,), which will e mjor focus of this review. Feed-forwrd inhiition commonly occurs in severl regions of the nervous system, including neocorticl, hippocmpl, sl gngli nd thlmic networks. We will discuss how chnges in feed-forwrd inhiition in different circuits cn cuse norml circuit dynmics tht underlie epileptic seizures. Feed-forwrd inhiition in neocortex nd hippocmpus. Incoming sensory signls trveling from the periphery to the cortex rise from the thlmus in the form of glutmtergic excittion tht is lrgely focused on the sensory receptive zone in the corticl lyer 4 (ref. 3). In turn, intrcorticl circuits re composed lrgely of excittory neurons tht re recurrently connected 4,5. These neurons mplify nd process incoming signls y propgting through cnonicl microcircuit to superficil nd then deeper corticl lyers. Although incoming sensory signls re excittory, prominent feture of neocorticl microcircuits is feedforwrd inhiition medited predominntly y fst-spiking (FS) sket cells contining the clcium-inding protein prvlumin (prv). Thus, incoming sensory signls directly nd potently excite prv cells in lyer 4, cusing them to fire nd relese the inhiitory neurotrnsmitter GABA onto excittory neurons in this lyer. This cuses powerful feed-forwrd inhiition tht sets rief window for temporl synptic integrtion in which spikes cn e generted 6, nd n overll limit for overexcittion in the neocortex 5 8. Similr circuitry exists in the other corticl regions, including hippocmpl dentte gyrus 9. Notly, individul prv cells hve potent output, minly onto cell odies nd proximl dendrites, through convergent input to individul pyrmidl cells 10,11. This feture positions prv cells to powerfully suppress output of pyrmidl nd other principl cells. Note tht, lthough feed- forwrd inhiition generlly suppresses ctivity, under some conditions, feed-forwrd ctivtion of inhiitory neurons, especilly Chndelier cells, cn enhnce network output 12. Recent findings hve demonstrted connectivity rules tht dd level of complexity to feed-forwrd inhiitory circuits. Accordingly, prv sket cells in the CA1 region of hippocmpus do not indiscrimintely trget ll CA1 pyrmidl neurons in the domin of their xonl ror, ut specificlly trget susets of pyrmidl neurons with their own specific output projections 13. Thus, this represents nother potentil choke point, s trgeted excittion of relevnt prv cells tht suppress output to specific region could prevent propgtion to tht region. The powerful nture of feed-forwrd inhiition in thlmocorticl (nd other) circuits results from severl fctors, including lrger convergence of single-fferent thlmocorticl xons onto individul prv-inhiitory cells tht relily generte spikes 8,14 16, divergence of output from such prv cells 17,18 nd the strength of unitry connections from individul prv cells 8,11. These oservtions support the hypothesis tht the nervous system opertionlly requires dequte feed-forwrd inhiition, nd filure of this key microcircuit leds to over-excittion of corticl networks nd seizures. This hypothesis is supported y evidence in severl models of epilepsy, including those induced y neontl corticl freeze lesions tht result in focl corticl dysplsi 7 nd in the strgzer 19, tottering 20 nd Gri4 / (ref. 21) models of generlized-sence epilepsy. Losing feed-forwrd inhiition is consistent with the dormnt sketcell hypothesis of epilepsy 22,23 : inhiitory neurons would lose so much connectivity tht they would egin to fil in their necessry role of providing timely feed-forwrd inhiition. Although the dormnt sket cell theory considers oth feed-forwrd nd feed-ck inhiition (discussed elow), the former hs often een shown to hve mjor role in studies with in vitro slice or whole hippocmpl models tht cutely induce epileptiform ctivity with chemoconvulsnts Indeed, one group 24 found tht prv cells re primrily involved in feed-forwrd inhiition, much more so thn somtosttin-positive (SOM) interneurons, which re the second-lrgest popultion of interneurons. The ltter pper to e more responsile for feed-ck inhiition. The dormnt sket-cell hypothesis hs een controversil in terms of the ctul circuit chnges tht might cuse dormncy; however, it remins criticl, s loss of Figure 2 Feed-forwrd inhiition in corticl nd thlmic microcircuits. () Extrinsic excittory projections from regions outside of locl corticl networks recruit feed-forwrd inhiition. Corticl inter-rel or thlmic inputs to the cortex result in stronger ctivtion of FS prv cells thn excittory stellte nd pyrmidl cells, therey cusing roust feed-forwrd inhiition of excittory cells. In the cse of loss of this feed-forwrd inhiition (erser*), thlmic inputs to the cortex recruit epileptiform ctivity in neocorticl microgyrus model of focl neocorticl epilepsy (ottom multi-unit nd locl field recordings 7 ). () Excittory inputs from the cortex to the thlmus results in stronger ctivtion of the inhiitory interneurons, which cuses strong feed-forwrd inhiition of rely excittory neurons. Loss of feed-forwrd inhiition (erser*) hs een implicted in the Gri4 / mouse model of sence epilepsy (multi-unit recordings 21 ). Blck circles indicte electricl stimultion of excittory fferents., cortex; PV, prvlumin-positive interneuron;, pyrmidl neuron; RT, reticulr thlmic neuron; St, stellte;, thlmocorticl neuron. Purple nd red represent excittory glutmtergic nd inhiitory GABAergic neurons, respectively. Feed-forwrd inhiition in cererl cortex PV St Microgyri 100 ms Feed-forwrd inhiition in thlmus Wild type RT Gri4 / 20 μv 100 μv 1 s 352 volume 18 numer 3 march 2015 nture neuroscience

3 feed-forwrd inhiition, with its powerful effects on the function of locl excittory neurons, cuses potent dysfunction of circuits. Notly, feed-forwrd inhiition hs een shown to prevent seizures from developing. Indeed, selectively impiring C 2+ chnnels in neocorticl prv interneurons 27, which would cuse loss of feed-forwrd inhiition, produces generlized-sence seizures. Similrly, specific reduction of the intrinsic excitility or synptic excittion of prv inhiitory interneurons, ut not of excittory cells, decreses feed-forwrd inhiition. In recent studies, reduced function of N v 1.1 sodium chnnels in prv FS interneurons ws implicted in epileptic seizures in mouse model of severe Drvet syndrome In ddition, deficits in N v 1.1 in prv neurons contriute to epileptiform hippocmpl ctivity in mouse models of fmilil Alzheimer s disese. Moreover, overexpressing N v 1.1 reduces epileptiform ctivity 31. By considering how prv cells ffect feed-forwrd inhiition, we propose tht rescuing hypofunctionl inhiition could prevent seizures y restoring feed-forwrd inhiition. Cn feed-forwrd inhiition regulte seizure propgtion over long distnces? According to studies with novel in vitro preprtions tht retin cllosl or commissurl connections, it cn. For exmple, in cllosum-intct ilterl neocorticl slice preprtion 32, chemiclly induced epileptiform ctivity leds minly to feed-forwrd inhiition in the contrlterl cortex. Similr effects occurred in ilterl-intct hippocmpl preprtions, especilly in the erly phse of seizure induction in which interictl spikes were most prominent 26. Thus, prominent phsic inhiition from fr cn signl n impending seizure. Feed-forwrd inhiition lso criticlly regultes the dynmics of the hippocmpl network, s shown in models of temporl loe epilepsy (TLE). In this network, the feedforwrd excittory tri-synptic loop is generlly considered to e responsile for propgtion from entorhinl cortex to dentte gyrus to CA3 to CA1; however, the hippocmpl network contins other pthwys tht my e importnt for seizure genesis nd/or propgtion. For exmple, in ddition to the entorhinl projection to dentte, there is lso projection directly to CA1 through the temporommonic pthwy. Losing feed-forwrd inhiition in this pthwy occurs in the pilocrpine model of TLE s result of severl fctors, including cell loss in superficil neurons in lyer 3 of the entorhinl cortex 33, which project to hippocmpl CA1 (ref. 34); loss of strtum oriens-lcunosum moleculre (O-LM) interneurons 35, which, in ddition to their mjor role in feed-ck inhiition, lso medite feed-forwrd inhiition in the tempero-mmonic pthwy 36 ; nd distl dendritic inhiitory denervtion of hippocmpl CA1 cells, region preferentilly regulted y O-LM interneurons 37,38. Comining these processes would produce loss of feed-forwrd inhiition from the entorhinl cortex to CA1. This hypothesis is consistent with results of voltge-imging study in which entorhinl stimultion mssively ctivted the pthologicl network in CA1 hippocmpus of post-pilocrpine epileptic nimls 39. Notly, surviving O-LM cells in CA1 send errnt fiers into dentte gyrus, which my, t lest prtilly, compenste for the loss of locl dentte inhiitory cells 40. Feed-forwrd inhiition cn lso e relevnt to intr-rel corticl excittion. It is lrgely responsile for surround inhiition, which ws documented decdes go in pioneering studies of cute neocorticl or hippocmpl seizures in felines 41,42. Recently, oth feed-forwrd nd surround inhiition hve een investigted with opticl nd electrophysiologicl methods to study the spred of seizures from focl zone tht initites epileptic seizures, the ictogenic zone. These results, otined lrgely in rodent models in which epileptic seizures were induced y chemoconvulsnts, show tht the erliest forms of periictl synptic ctivity re multiphsic, repetitive nd potent inhiitory signls. This erly ctivity is ssocited with norml (non-ictl) ckground ehvior in the network, ut is followed y sudden collpse of inhiition, such tht strong excittory signls dominte individul cellulr responses. As result, these signls produce precipitous steplike wves of locl excittion t the network level, s oserved with C 2+ imging 43. This cycle then repets to propgte seizure ctivity to the next microcircuit. Recently, nlogous neurl ctivities hve een reveled from intr-opertive intrcrnil electricl recordings otined from the cererl cortex of epilepsy ptients eing evluted for neurosurgicl resections 44. These recordings suggest tht during clinicl seizures, feed-forwrd inhiition fils through mechnisms similr to those oserved in experimentl nimls. Feed-forwrd inhiition in thlmus. Circuit motifs differ etween rin regions, especilly etween corticl nd sucorticl microcircuits. The thlmus, s sensory rely sttion, shpes incoming peripherl informtion through three inhiitory pthwys: 1) feed-forwrd dendro-dendritic inhiition medited y locl circuit interneurons tht sculpt pckets of primry fferent signls to dely firing 45, 2) direct feed-ck inhiition driven y triggering thlmocorticl () drive of inhiitory thlmic reticulr (RT) neurons, nd 3) inhiition vi the RT nucleus triggered y corticl feedck. The ltter form cn e confusing, s recurring excittory signls from cortex to thlmus would, from systems perspective, e considered feedck. Yet, from microcircuit perspective, output from cortex triggers feed-forwrd inhiition, s the mjor effect of corticl output is preferred recruitment of inhiitory cells in the RT nucleus 21,46. Thus, RT cells provide powerful inhiitory output onto excittory rely cells. Recent studies hve suggested tht loss of feed-forwrd inhiition in the cortico-thlmic pthwy cn e epileptogenic. For exmple, studies reveled tht inhiitory RT neurons lose AMPA-medited excittion in two genetic models of generlized-sence epilepsy: strgzer nd Gri4 / mice 21,47,48. In the ltter model, the synptic defects in the cortico-thlmic microcircuit were deconstructed with optogenetics, promising new pproch to studying epileptogenetic pthwys. This pproch reveled how loss of specific microcircuit component synptic excittory drive from neocortex onto inhiitory RT cells cn cuse deficit in feed-forwrd, ut not feed-ck, inhiition 21 (Fig. 2). These findings suggest tht even though corticl efferents re lrgely, if not exclusively, excittory, their primry effects on thlmic ctivity cn e inhiitory (for discussion of the potentil physiologicl roles for such feed-forwrd inhiition, see ref. 49). These results further suggest tht specificlly restoring excittory inputs from the cortex onto RT cells would rescue feed-forwrd inhiition nd suppress sence seizures tht would otherwise develop in the thlmocorticl network. Feed-forwrd inhiition: potentil trget of nti-epileptic drugs? Feed-forwrd inhiition is criticl for norml circuit function, yet is lso prdoxiclly frgile ecuse of severl fctors, including intrcellulr Cl ccumultion, GABA depletion nd presynptic inhiition Altering these fctors with drugs my crete restortive tretments ginst epilepsy. Furthermore, if loss of feed-forwrd inhiition is cuse of epilepsy, then nti-epileptic drugs (AEDs) should in principle re-estlish it, nd in no cse should they suppress it. However, severl AEDs, including phenytoin, crmzepine 54,55 nd lmotrigine 56, my work through mechnism tht locks N + chnnels, especilly in the context of ction potentils tht fire t high frequency. Prv cells, which lrgely medite feed-forwrd inhiition nd fire t high frequencies, my e susceptile to reduced firing y AEDs. Thus, AEDs could potentilly worsen seizures. To resolve this prdox, study recently ddressed the effects of N + chnnel lockers (for exmple, the nti-convulsnt drugs crmzepine, phenytoin nture neuroscience volume 18 numer 3 march

4 CA1 Feed-ck inhiition in hippocmpus SOM Feed-ck inhiition in thlmus RT Figure 3 Feed-ck inhiition in corticl nd thlmic microcircuits. () In the cortex, inhiitory SOM interneurons provide feed-ck inhiition to pyrmidl neurons tht excite them. Loss of this inhiition (erser*) hs een implicted in temporl loe epilepsy (TLE) 37. () In the somtosensory thlmus, inhiitory interneurons provide roust feed-ck inhiition to neurons tht excite them. Incresing this feed-ck inhiition (dumell weight *) y Zolpidem 70 or y clonzepm in α3h126r mice (not shown 69 ), which specificlly ffects RT-, ut not RT-RT connections, increses the strength of epileptiform oscilltions. SOM, somtosttinpositive. Purple nd red represent excittory glutmtergic nd inhiitory GABAergic neurons, respectively. Control TLE 10 mv 50 ms Control 500 nm Zolpidem 0.5 s nd lmotrigine) on different cell types. These compounds specificlly reduced repetitive firing in pyrmidl neurons, ut not in FS or other interneurons 57. The AEDs lso did not ffect recruitment of inhiition during repetitive ctivity. Thus, AEDs reduce ction potentil firing primrily in excittory neurons nd spre interneurons to mintin feed-forwrd nd other forms of inhiition. To conclude, the ntomicl connectivity nd functionl fetures of prv sket cells in cortex nd hippocmpus nd prv RT cells in the thlmus enle them to serve s centrl plyers in feed-forwrd inhiition. Furthermore, this inhiition is well-positioned to prevent epileptic ctivity from ridging etween microcircuits, nd its filure could redily propgte seizures. Thus, meditors of feed-forwrd inhiition, minly prv cells, could serve s potentil seizure choke points. Feed-ck inhiition In contrst with feed-forwrd inhiition, which is microcircuit motif engged y extrinsic sources, feed-ck inhiition generlly results from excittion in locl circuit elements (Fig. 3,). Similr to feedforwrd inhiition, feed-ck inhiition is common theme in cererl circuits. Although different clsses of inhiitory cells cn medite oth forms of inhiition, their reltive roles differ. Indeed, the prv cells descried ove pper to hve mjor role in feed-forwrd inhiition, wheres second mjor clss of inhiitory cells, SOM-contining interneurons, ppers to e more importnt in feed-ck inhiition. Although diverse suclsses of SOM cells cn e involved in epilepsy, we will focus our discussion minly on one suclss of SOM cells, Mrtinotti neurons, which trget distl dendrites of pyrmidl neurons 10,58,59. Compred with prv-medited inhiition, Mrtinottimedited inhiition is weker t seline ecuse postsynptic cells hve fewer synpses 11. However, Mrtinotti-dependent inhiition is progressively recruited y simultneous repetitive ctivity in multiple presynptic pyrmidl cells, s would hppen, for exmple, during intense ctivtion of locl microcircuits in seizures. Such recruitment results from fcilitting short-term synpses of oth the excittory inputs onto nd the inhiitory outputs from neocorticl Mrtinotti cells nd relted neurons of the hippocmpus In contrst, inhiition from prv sket cells is initilly roust ecuse of convergent input coupled with high-proility sites of relese onto pyrmidl cells. However, s result of short-term synptic depression, the efficcy of prv-medited inhiition rpidly drops during repetitive ctivtion 61. The progressive nture of Mrtinotti cell recruitment could e importnt for dmpening ctivity to loclly suppress seizures in the microcircuit. Consistent with this, mice deficient in the trnscription fctor DLX1 show reduced SOM cells nd mild epilepsy phenotype 63. Furthermore, in murine model of Drvet syndrome, SOM-medited inhiition is lso reduced 28. In ddition to SOM nd Mrtinotti cells, other neurons my contriute to feed-ck inhiition in epileptic microcircuits. For exmple, neocorticl chndelier cells, which trget the initil xon segments of pyrmidl neurons, my prevent hyperexcittion relted to epilepsy. In n in vivo study tht exmined the spontneous nd whisker-evoked ctivity of vriety of neuronl types in the rrel cortex, chndelier cells only responded wekly to whisker stimultion; only smll synptic potentils were oserved nd they rrely evoked ction potentils 64. However, disinhiition induced y locl corticl ppliction of the GABA-receptor ntgonist icuculline cused 20-fold increse in the spontneous firing rte of chndelier cells, which exceeded tht of ny other cells recorded. This finding suggests tht chndelier cells my e specificlly recruited y epileptic ctivity nd tht, y vetoing spike output vi shut-down of pyrmidl cell xons, my serve s microcircuit emergency rke. Although the specific excittory versus inhiitory effects of ctivting chndelier cells remin controversil 12,65 67, their ctivtion potentilly represents nother seizure choke point. Another exmple of the role of feed-ck inhiition in epilepsy comes from studies of thlmocorticl circuits primrily implicted in generlized-sence epilepsy. Here, feed-ck inhiition hs powerful seizure-promoting role, especilly in the thlmus. The thlmic network is composed of topogrphiclly relted, reciproclly connected inhiitory neurons in RT nd excittory cells locted in specific rely nuclei in dorsl thlmus 68 (Fig. 3). Activity of the excittory cells ctivtes synpses of RT neurons, cusing recurrent feed-ck inhiition in the sme cells. Such inhiition promotes ctivity of the oscilltory network in the thlmus, s cells exhiit form of prdoxicl ctivtion: they fire post-inhiitory reound ursts of ction potentils when strongly inhiited y synchronized output of RT neurons. At the microcircuit level, enhncing feed-ck inhiition with phrmcologicl interventions, such s those tht lock uptke of the inhiitory neurotrnsmitter GABA or phrmcologicl tretments tht specificlly trget RT- synpses, excerte epileptiform ctivity in vitro 69,70 (Fig. 3) nd worsen generlized-sence seizures in epilepsy ptients 71. In the thlmus, -RT- feed-ck inhiition cn promote seizure responses, wheres, in the cortex, feed-ck inhiition lrgely suppresses seizure ctivities. Thus, cution is required when interpreting results from glol gene knockout models tht generlly ffect microcircuits, such s those tht enhnce feed-ck inhiition. Similrly, tretments tht nonspecificlly trget feed-ck inhiition through the rin might not only e ineffective, ut might lso excerte seizures. To conclude, feed-ck inhiition cn engge specific microcircuits to either stimulte or inhiit seizure ctivity. Accordingly, we need to dissect relevnt microcircuits involved in ictogenesis to identify specific seizure choke points in different types of epilepsies. 354 volume 18 numer 3 march 2015 nture neuroscience

5 Counter inhiition in hippocmpus PV - - PV Counter inhiition in thlmus - RT RT - Figure 4 Counter-inhiition in hippocmpl nd thlmic microcircuits. () Inhiition etween FS prv cells in the hippocmpus cn enhnce gmm rhythmicity 81. Incresing this inhiition (weight*) hs een suggested to enhnce network synchrony ssocited with epilepsy. () Inhiition etween RT neurons in the thlmus desynchronizes the thlmic network oscilltions etween nd RT cells. Loss of RT-RT counter-inhiition (erser*) in GABA A receptor β3 suunit knockout mouse (β 3 / ) enhnces intr-thlmic network synchrony nd hs een implicted in epilepsy 87. Purple nd red represent excittory glutmtergic nd inhiitory GABAergic neurons, respectively. 50 ms 10 mv β 3 +/+ β 3 / 200 ms 100 μv Counter-inhiition The nervous system mkes its own, sometimes inscrutle, rules out the type nd strength of connections mde y ny individul cell type. In some cses, the synptic output of prticulr neuronl clss is quite promiscuous, s it couples indiscrimintely to ny nery neurons tht fll in its rnge of efferent xonl output 72 ; however, in other cses, it exclusively trgets either neurons of its own or other suclsses 73. Inhiitory neurons hve unique connectivity rules tht seem to tke this ide to the extreme. In ddition to their potent inhiitory output to pyrmidl neurons, prv sket cells form powerful utptic connections (tht is, they synpse onto themselves) 74,75, reltively rre form of connectivity in the nervous system. Along these lines, mny clsses of inhiitory interneurons mke chemicl nd/or electricl synptic connections with other interneurons in or outside their own clss 72,76,77, nd some inhiitory cell clsses (in corticl lyer I nd/or expressing the peptide vsoctive intestinl peptide, VIP) hve een shown to specificlly medite disinhiitory effects through inhiition of SOM nd prv cells Thus, stimultion of given set of inhiitory neurons could cuse specific disinhiitory effect, perhps promoting overexcittion, wheres inhiition of lyer I/VIP cells might produce n increse in SOM/prv output nd result in seizure choke point. Given the diversity of inhiitory motifs in microcircuits descried so fr, locking one of these motifs could hve disprte nd, perhps, opposite consequences on the overll function of microcircuits. Thus, counter-inhiition, inhiition of inhiition (Fig. 4,), is key concept in epileptic microcircuits. For exmple, counter-inhiition of prv sket cells my lrgely suppress feed-forwrd inhiition (motif 1) nd promote seizure propgtion etween regions, wheres counterinhiition of Mrtinotti cells my promote locl ictogenesis through loss of the progressively ctivted feed-ck circuit 60,62. Here we focus on one type of counter-inhiition: etween cells of the sme inhiitory clss. Counter-inhiition in neocortex nd hippocmpus. Counterinhiition cn promote ctivity through severl mechnisms. First, mong inhiitory cells, counter-inhiition cn disinhiit downstrem excittory cells, leding to generl increse in firing. Alterntively, it cn promote oscilltory ctivity in reciproclly connected networks. For exmple, synptic inhiition etween prv FS cells cn promote oscilltory output from microcircuits to produce gmm-frequency oscilltions 81. Such gmm- nd relted higher frequency oscilltions hve een implicted in ictogenesis in limic epilepsy 82 (Fig. 4). Counter-inhiition in thlmus. Counter-inhiition ffects thlmic function nd hs een implicted in ictogenesis in sence epilepsy. In thlmic microcircuits, RT neurons medite feed-forwrd nd feed-ck inhiition (s descried ove). In ddition, RT neurons re loclly interconnected y oth chemicl-inhiitory 83 nd electricl synpses 83,84. Chemicl inhiition etween RT cells is potent nd chrcterized y long-lsting synptic responses 85, nd cn limit the synchronous ctivtion of RT cells during epileptiform oscilltory responses in the network 86. Thus, specific loss of RT-RT counterinhiition y deleting criticl, nucleus-specific GABA A -receptor β3 suunit is ssocited with enhnced emergent hypersynchrony nd the development of epilepsy 87 (Fig. 4). Accordingly, trgeting hypersynchrony nd epilepsy in thlmic networks with phrmcotherpies will need to cuse greter net effect on RT-RT inhiition (nti-oscilltory) versus -RT- feed-ck inhiition (pro-oscilltory) 69. Indeed, the nti-epileptic drug clonzpm decreses the output of RT neurons to cells y enhncing RT-RT counter-inhiition 88. Thus, in contrst with the generlly suppressive effects on trget excittory cells descried ove for feed-ck nd feed-forwrd inhiition, counter-inhiition cn promote or reorgnize the excittory ctivity of microcircuits, respectively. These effects cn occur either through disinhiition or entrinment of recurrent inhiitory networks tht produce periodic-phsed synptic inhiition to control the timing of excittory cells. Recurrent excittion This recurrent excittion microcircuit motif (Fig. 5,) flls well within the context of the excittion nd inhiition discussions of epileptogenic mechnisms, nd for good reson. Recurrent excittion is enhnced in most experimentl epilepsies. However, modern pproches re now promoting identifiction of specific, nd sometimes de novo, chnges in excittory circuits. One powerful pproch is photo-stimultion, often with photo-lile lignds such s cged-glutmte 89. With this pproch, originlly reported over decde go, light cn e foclly delivered to specific loctions in rin circuit, most commonly in n cute rin slice. This light ctivtes neurons in tht region nd genertes synptic excittory signls in neurons postsynptic to the stimulted cells. This pproch showed tht recurrent excittion in the dentte gyrus commonly occurred in limic epilepsy model 90. More recently, this pproch reveled intricte chnges in dentte connectivity, with notle increses in inputs to dentte gyrus grnule cells from not only other grnule cells, ut lso hilr excittory neurons nd CA3 pyrmidl neurons 91 (Fig. 5). Such chnges cn crete strong sis for hyperconnected, epileptic network, especilly if the reorgniztion follows the principles of hu-cell connectivity, in which smll numer of well-connected neurons help develop complex network ctivity such s seizures 92. In neocortex, recurrent excittory connections re enhnced following corticl injury nd re notly precise. For exmple, in the isolted corticl sl, which produces epileptogenic insult (Fig. 5), enhnced connectivity is restricted to infrgrnulr lyers, especilly lyer 5 (ref. 93); however, in model of focl corticl dysplsi, enhnced connectivity to lyer 5 cells is seen from oth infr nd supr-grnulr regions 94. These findings suggest tht lesion-specific reorgniztion occurs in different injury models. nture neuroscience volume 18 numer 3 march

6 Recurrent excittion in postlesionl cortex Non-epileptiform Epileptiform Interventions tht counterct or reverse such enhnced reorgniztion of excittory microcircuits my yield novel therpeutic pproches. Notly, these pproches will e most effective if they specificlly trget mldptive reorgniztions in excittory networks nd mintin norml function of recurrent excittory networks. Microcircuit interctions Thus fr, we hve reviewed the properties of isolted microcircuits relevnt to ictogenesis, including the importnt fetures of connection sign (inhiitory nd excittory), sptil pttern (convergence nd divergence) nd trget region (som, dendrite nd xon). These fetures re ll reltively sttic in microcircuits, yet mny synptic nd cellulr components of the circuits cn e dynmiclly modulted to crete stle microcircuit tht could, under the right (or wrong!) conditions, progressively shift to n ictogenic form. Furthermore, s indicted t the outset of this review, individul microcircuits do not exist in isoltion, nd epilepsy results from propgtion of ictl ctivity through the distriuted microcircuits. We suggested the ide tht n imlnce etween diverse microcircuit motifs, such s etween feed-ck nd feed-forwrd inhiition, cn e ictogenic. As mentioned ove with regrds to Gri4 / mice, sence epilepsy results from lck of feedforwrd, ut unffected feed-ck, inhiition. In this cse, specific defect t the cortico-rt synpse results in lck of cortico-rt- feedforwrd inhiition, which cuses norml recruitment of cells y fferent excittory inputs (tht is, multiple cells re concurrently ctivted y corticl output), wheres the intct -RT pthwy results in powerful -RT- synchronized feed-ck inhiition. Thus, n imlnce etween feed-forwrd nd feed-ck inhiition enles norml excittory inputs to recruit seizures 21. To conclude, this cse, in prticulr, supports the emerging concept tht the field needs to expnd eyond the historicl view tht epilepsy simply results from n imlnce etween excittion nd inhiition nd consider the possiility tht epilepsy cn lso result from n imlnce etween different microcircuit motifs. Dynmics in microcircuits As indicted ove, synptic connections re considerly heterogeneous, not only in trgets nd connection strength, ut lso in short-term dynmics. For exmple, sket-cell output synpses show short-term depression nd lose efficcy over time, nd SOM nd Mrtinotti cells show the opposite y ugmenting synpses tht increse in efficcy over time. Such dynmic chnges will inevitly lter the lnce etween different forms of inhiition. Thus, the normlly high rtio of inhiitory output of sket cells (minly prv to somtic trgets) to Mrtinotti nd relted cells (SOM to dendritic trgets) oserved during physiologicl ctivity will e replced y n inverted rtio in Control 0.5 mv GC 50 ms Hilus Recurrent excittion in postlesionl hippocmpus GC TLE pa Figure 5 Recurrent excittion in cortex nd hippocmpus. () Recurrent excittion etween pyrmidl excittory cells (weights*) increses fter neocorticl lesions nd hs een implicted in polysynptic epileptiform ctivities in the undercut model of focl neocorticl epilepsy 107. Bottom trces, locl recordings of epileptiform field potentils from the injured neocortex evoked y electricl stimultion (lck circle). () Ectopic recurrent excittion (weight*) etween presynptic excittory neurons in dentte, hilus, nd CA3 nd postsynptic grnule cells in the hippocmpus develops in the pilocrpine model of temporl loe epilepsy. Bottom, connectivity mps sed on glutmte photo-uncging evoked excittory postsynptic currents in slices from control nd epileptic (TLE) mice 91. Purple represents excittory glutmtergic GABAergic neurons. which Mrtinotti cell output predomintes 61. This effect my suppress norml ctivity in n ictogenic microcircuit, ut leve tht sme microcircuit vulnerle to dditionl extrinsic ictogenic signls cused y loss of feed-forwrd inhiition. Externl influences on microcircuits Activity cn e propgted etween microcircuits through efferent projections to circuit elements outside of the microcircuit. Indeed, longrnge excittory projections connect distl cererl res. For exmple, the corpus cllosum is composed lrgely of xons of excittory corticl neurons 95, nd this mjor commissurl trct is responsile, in lrge prt, for propgtion of seizures 96. In recent work, certin clsses of inhiitory neurons were found to lso mke long-rnge connections tht would influence locl nd glol epileptic networks. These findings hve recently een reviewed elsewhere 97 nd will not e further discussed here, except to highlight tht this theme is emerging with potentil relevnce to the motifs descried ove nd their ictl choke points. As with intr-hemispheric cererocorticl networks, corticothlmocorticl networks re connected through long-rnge, reciprocl excittory projections. Sensory regions of dorsl thlmic nuclei re composed lrgely of excittory feed-forwrd excittory neurons tht trnsfer peripherl sensory informtion to the cortex vi projections primrily to corticl lyer 4. There, ctivity reverertes nd propgtes etween corticl lyers 4 to ultimtely end up in deep corticl lyers, including lyer 6. Lyer 6 neurons then emit xons ck to thlmus to re-excite the neurons. In sensory thlmus nd cortex, this synptic reltionship is topogrphic in oth directions, leding to highly loclized, ut long-loop, excittory recurrent network. Interposed on this, nd indeed emedded in it, is the intrthlmic loop etween neurons nd inhiitory RT neurons. As we descried ove, this emedded reciprocl reltionship etween circuits is kept in check y powerful feed-forwrd inhiition from the cortex tht prevents significnt excittion of rely neurons tht might led to runwy excittion nd seizures. An dditionl considertion regrding extrinsic influences on microcircuits is the effect of neuromodultory pthwys, which cn selectively nd specificlly ct on individul microcircuit components. For exmple, cholinergic modultion disprtely inhiits sket cells nd ctivtes presumed SOM cells 10. Of note, recent studies hve shown tht suset of nrrow spiking neurons, presumed sket cells, is negtively modulted y ttendnce to visul tsk. This finding suggests tht ttentionl sttes cn led to disinhiition through specific chnges in inhiitory microcircuits 98. The potentil relevnce of such chnges to epilepsy remins to e studied. Circuit therpy: where re the choke points? Although the process of developing epilepsy, epileptogenesis, likely entils multiple dptive nd mldptive circuit chnges, we hve ddressed severl simple microcircuit motifs in which dysfunction in one element (for exmple, synpse or neuron), either through 356 volume 18 numer 3 march 2015 nture neuroscience

7 Figure 6 Circuit therpy: focus on choke points. () The thlmus is choke point for epileptic seizures in post-stroke epilepsy 99. Note tht the choke point (yellow flsh, thlmus) is remote from the initil dysfunction (red flsh), which is stroke in the cererl cortex. () The STN is n efficient choke point for pthologicl circuit oscilltions in Prkinson s disese. Note tht the choke point (yellow flsh, STN) is remote from the initil dysfunction (red flsh), which results from degenertion of dopminergic cells (dopmine) projecting from the sustnti nigr compct (SNC) to stritum. (c) Contrlterl hippocmpus is choke point for controlling ipsilterl hippocmpl epileptic ctivity 100. (d) STN nd sustnti nigr prs reticult (SNR) re choke points for spike-nd-wve dischrges ssocited with sence epilepsy nd generted in somtosensory cortex 108. Blck oscilltions indicte pthologicl oscilltions, the red flsh indictes initil injury or insult, nd the yellow nd lue flshes indicte choke points for pthologicl network oscilltions. GPe, externl glous pllidus. Purple cells nd projections re excittory glutmtergic, nd red cells nd projections re inhiitory GABAergic. gin or loss of function (for exmple, chnge in synptic strength or intrinsic excitility), cn effectively entrin locl network ctivity. The uild-up of such locl ctivity to the point of inititing seizure is n ictogenesis. Thus, in ech of the four different cses of mldptive circuit motifs, restortive tretments tht would reverse or counterct the specific dysfunction (or perhps prevent the dynmic recruitment of tht dysfunctionl element during ictogenesis) could crete n effective nti-seizure therpy. By extending this pproch, some regions other thn the point of mximl dysfunction might e trgeted (Fig. 6 d). Distl trgeting might e more efficient ecuse the distl sites re either criticl in glol ictogenesis nd/or re more sptilly restricted, nd therefore esier to mximlly trget. If the cells in distl sites re only modestly involved in glol ictogenesis, then reducing the ctivity of only some of them will not e effective. However, if they re concentrted in region such tht the ulk of relevnt cells in the distl sunetwork cn e effectively trgeted, then gret efficcy would e gined. For exmple, rt model of corticl photothromotic stroke developed epilepsy over time (Fig. 6). Here, specificlly inhiiting the portion of thlmus projecting to the surviving peri-infrct cortex ws sufficient to ort, in rel-time, utomticlly detected seizures 99. Becuse of extensive long recurrent excittory connections with cortex, these results suggest tht thlmus could e n importnt trget in epilepsies resulting from corticl lesions other thn stroke. Severl dditionl exmples of loclized, off-site seizure control re evident nd further support the ide tht remotely regulting seizures might crete generlly useful concept regrding ictogenic choke points. For exmple, in model of limic epilepsy cused y unilterl intrhippocmpl injection of the excitotoxin kinic cid, optogenetic excittion of inhiitory cells of either the primry ipsilterl epileptogenic zone or in the contrlterl hippocmpus reduced seizures 100 (Fig. 6c). In nother exmple of off-site control, this sme group hs shown tht optogenetic ctivtion of cereellr Purkinje neurons suppresses seizures in this niml model of epilepsy 101. In ddition, experimentl seizures induced y either electricl or chemicl stimulnts re strongly suppressed y loclly inhiiting the sustnti nigr 102. Thus, trgeting such sucorticl Cererl cortex Thlmus nrt c Thlmic choke point in post-stroke epilepsy Injury CT VPL VPM Peri-stroke CT Contrlterl hippocmpus Insult Thlmus d Cererl cortex Cererl cortex Thlmus STN STN structures, such s the thlmus or sustnti nigr, remote from the initil corticl dysfunction, might hve mjor dvntges. For instnce, trgeting the thlmus in rel time would e less deleterious thn trgeting the eloquent cortex. We propose tht the thlmus could e choke point in epileptic circuits in the sme wy tht the suthlmus (STN) is choke point for norml circuit dynmics in Prkinson s disese. Indeed, the concept of circuit motif choke points cn e rodly pplied to nervous system disorders. In the cse of Prkinson s disese, the initil dysfunction results from the degenertion of neurons in the sustnti nigr prs compct nd, therefore, is remote from the STN. However, trgeting the STN is the mjor therpy used in Prkinsonin ptients. Indeed, the STN is choke point of norml circuits in Prkinson s disese ecuse of its key loction in the circuit, even though the initil dysfunction is remote (Fig. 6) 103. Of note, high-frequency stimultion of STN or inhiition of sustnti nigr prs reticult 104,105 lso strongly suppresses seizures in GAERS 106 model of generlized-sence epilepsy further supporting the concept of distl epileptic choke points. Conclusions Although we need to identify the focus of the initil dysfunction, we lso need to look for potentil control or choke points tht re remote nd could e distnt from the focus of the initil dysfunction. Thus, y scnning regions outside tht of the initil insult, we my find foci fr from wht hs historiclly een considered the focus nd, in so doing, my find unique opportunities for effective therpies tht trget these circuits. ACKNOWLEDGMENTS We would like to thnk C. Mkinson for criticl comments. This work is supported y the US Ntionl Institutes of Helth nd the Ntionl Institute of Neurologicl Disorders nd Stroke, nd Citizens United Aginst Epilepsy. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. CT Suthlmic choke point in Prkinson s disese Focus Reprints nd permissions informtion is ville online t reprints/index.html. GPe GPe SNR SNR Bsl gngli choke points in sence epilepsy Stritum Dopmine Injury Stritum SNC nture neuroscience volume 18 numer 3 march

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