Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury

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1 a r t i l e s Closed-loop optogeneti ontrol of thalamus as a tool for interrupting seizures after ortial injury Jeanne T Paz 1, Thomas J Davidson 2, Eri S Frehette 1, Bruno Delord 3, Isael Parada 1, Kathy Peng 1, Karl Deisseroth 2 & John R Huguenard Nature Ameria, In. All rights reserved. Cereroortial injuries suh as stroke are a major soure of disaility. Maladaptive oequenes an result from post-injury loal reorganization of ortial iruits. For example, epilepsy is a ommon sequela of ortial stroke, ut the mehanisms respoile for seizures following ortial injuries remain unknown. In addition to loal reorganization, long-range, extra-ortial onnetio might e ritial for seizure maintenane. In rats, we found that the thalamus, a struture that is remote from, ut onneted to, the injured ortex, was required to maintain ortial seizures. Thalamoortial neuro onneted to the injured epilepti ortex underwent hanges in HCN hannel expression and eame hyperexitale. Targeting these neuro with a losed-loop optogeneti strategy revealed that reduing their ativity in real-time was suffiient to immediately interrupt eletrographi and ehavioral seizures. This approah is of therapeuti interest for intratale epilepsy, as it spares ortial funtion etween seizures, in ontrast with existing treatments, suh as surgial lesioning or drugs. Despite the high prevalene of stroke and epilepsy, oth major soures of disaility 1 4, the underlying ellular and iruit mehanisms leading from stroke to epilepsy remain unknown, and injury-indued epilepsies often do not respond to existing treatments 3,5. Identifiation of the neural iruits involved in injury-indued epilepsy is neessary to pinpoint the exat nature of the ausally important alteratio in these iruits and to ontrol seizures with speifi interventio. Although altered short-range ortial iruit funtion is thought to e the primary ause of injury-indued epilepsies 6,7, the role of long-range onnetio to and from injured ortex to other rain regio in seizure maintenane has not een adequately studied. The ortex is intimately onneted with thalamus, and the ortiothalamo-ortial exitatory loop mediates network osillatio underlying epilepsies in man and in animal models 8. However, despite pioneering studies that have shown the reruitment of thalamus in ortial seizures 8 13, its role in seizure expression, partiularly in aquired epilepsies, remai unknown. The thalamus is omposed of oth exitatory and inhiitory neuro, and adjaent ortiothalami and thalamoortial glutamatergi axo. Thus, it is hallenging to seletively ativate thalamoortial or ortiothalami axo and it is impossile to ontrol their ativity in real time using traditional pharmaologial or eletrial manipulatio. We overame this hallenge using optogeneti methods 14 that allow for ontrol of a speifi ell type with high temporal preision. Although reent studies have used optogeneti tools to study seizure-like ativities in vitro 15,16, whether there is a ausal relatiohip etween the ativity of one ell type and the seizure and its disruption in real time has not yet een tested in ehaving animals. The peri-infart ortex is generally oidered to e the zone in whih seizures initiate 6,7, yet the extent of ortial damage preludes effetive antiepilepti targeting of the entire peri-infart hyperexitale ortex. In addition, targeting of suh exteive areas might disrupt funtion of eloquent ortex. We sought to determine, independent of where seizures initiate, whether the seondarily damaged thalamus is required to sustain post-stroke seizures and, if so, whether this foal region ould e readily targeted to stop seizures. We indued foal ortial strokes and loated and delineated potentially epilepti ellular and iruit hanges in thalamus in vitro. We then engineered an approah to speifially inhiit output from that thalami region to test whether it is ausally involved in seizure maintenane in vivo. For this purpose, we used an estalished model of ortial photothromosis that results in late epilepsy (>1 month) after stroke 17,18. This injury is assoiated with redued exitaility and exitation in the inhiitory retiular thalami neuro and retrograde ell death and gliosis speifially in ipsilateral ventroposterolateral thalami nuleus (VPL); these hanges are known to e omplete y the end of the first week and are sustained over months 19 (Supplementary Fig. 1a,). RESULTS Cortial stroke enhanes exitaility of thalamoortial ells Ipsilateral thalamoortial neuro showed inreased memrane intrii exitaility and enhaned rhythmogeni properties 7 14 d after foal ortial stroke. Speifially, input resistane in thalamoortial ells was inreased as a result of a redution in memrane 1 Department of Neurology and Neurologial Sienes, Stanford University Shool of Mediine, Stanford, California, USA. 2 Department of Bioengineering, Stanford University Shool of Mediine, Stanford, California, USA. 3 Ititut des Systèmes Intelligents et de Rootique, Centre National de Reherhe Sientifique, Unité Mixte de Reherhe 7222, Université Pierre et Marie Curie, Paris, Frane. Correspondene should e addressed to J.R.H. (john.huguenard@stanford.edu) or J.T.P. (jtpaz@stanford.edu). Reeived 1 Otoer; aepted 23 Otoer; pulished online 7 Novemer 212; doi:1.138/nn.3269 nature NEUROSCIENCE advane online puliation

2 a r t i l e s 212 Nature Ameria, In. All rights reserved. Figure 1 Cortial stroke results in enhaned intrii exitaility in thalamoortial neuro. (a) Confoal image of a horizontal thalami slie ipsilateral to the stroke (7 d post-stroke) with GFAP immunolaeling and thalami neuro laeled y intraellular injetion of ioytin (green, white arrows) during reordings. Pink traes show respoes to intraellular injetion of urrent pulses (from 2 pa to +8 pa) in thalamoortial ells from different thalami nulei ipsilateral to the infart. White traes show respoes from thalamoortial ells in ontrol rats using the same protools from orresponding nulei. (,) Representative voltage respoes to urrent injetio in thalamoortial ells from an injured and a ontrol rat. Note the enhaned intrii reound properties in injured ells (arrowheads, larger post-inhiitory after-depolarization, enhaned hyperpolarization-ativated sag and post-hyperpolarization urst). (d) Confoal images of representative ontrol and injured Bioytin 2 µm Bioytin 2 µm thalamoortial neuro (7 d post-stroke). (e) Passive memrane properties: apaitane (C m ), input resistane (R in ) and memrane time otant (τ m ) in thalamoortial ells lose to or far from (>2 µm) gliosis at 2 d, 7 14 d and >1 month post-stroke (data are presented as mean ± s.e.m.;, P >.1; *P <.5, one-way ANOVA). The numer of ells is indiated for eah group. Data are from 9 ontrol and 12 injured rats. AP, ation potential; IC, internal apsule; nrt, Po, VL, VPL, VPM: retiular, posterior, ventrolateral, ventroposterolateral and ventroposteromedial thalami nulei. area (Fig. 1), and the resting memrane potential was slightly depolarized (data not shown). Coistent with the inreased input resistane after injury, rheoase was redued, whereas the properties of individual ation potentials were not altered (Supplementary Tale 1). We oserved several alteratio in the iophysial properties of hyperpolarization-ativated yli nuleotide gated (HCN) hannel mediated urrent (I h ) in affeted thalamoortial ells (Fig. 2): faster ativation, depolarized half-ativation voltage (V 5% ), a I tail / I tail max e g (s) Ativation V h n = 1 ells V m () V m () I h ativation V h I h de-ativation 6 15 De-ativation I tail / I tail max (s) n = 6 ells V m () 1 ells 7 ells V m () 1 na a d () d IC +8 pa Bioytin GFAP 1 na (pa pf 1 ) 2 pa nrt I h deity 7 14 d V 5% *** * V = 95 V = 13 VL >1 month d >1 month f h 1 7 A = 6 D = 2.3 s D = 3.4 s A = 1.8 s VPL VPM e C m (pf) Po µm VPL 2 pa 34 AP +28 pa VPM * * 3 12 * d 7 14 d >1 month R in (MΩ) +8 pa 2 pa +8 pa 2 pa * VPL (gliosis) 2 pa 3 AP +16 pa 25. VPM (far from gliosis) 25. no hange in I h deity and a redued respoiveness of I h to AMP (data not shown). Changes in I h resulted, in part, from enhaned otitutive AMP signaling (data not shown) and a swith in predominant HCN suunits (Supplementary Fig. 2). Notaly, similar hanges in I h have also een linked to geneti epilepsies (for a review, see ref. 2). The inreased intrii exitaility was roust in ells lose to the injured glioti area (medial VPL, lateral ventroposteromedial thalami nuleus (VPM)), ut was not signifiant (P >.1) in thalami areas that were not affeted y ell death or gliosis, suh as medial VPM or ventrolateral thalami nuleus (Fig. 1a,e). Notaly, the hanges in intrii exitaility started in the first week after stroke, efore seizures, and persisted into the hroni post-stroke epilepti state (>1 month), suggesting that these hanges do not represent a traient, pre-apoptoti phenomenon d 7 14 d >1 month Far from inj. * m (MS) d 7 14 d >1 month Figure 2 Cortial stroke alters iophysial properties of I h in thalamoortial neuro. (a) Current respoes (top traes) to voltage steps (ottom traes) in representative thalamoortial ells from an injured and a ontrol rat. I h was measured at the tail urrent (arrowheads; see Online Methods). () The deity of I h at maximal ativation was similar in thalamoortial ells from injured and ontrol rats at oth 7 14 d and at >1 month post-stroke. () I h voltage-dependent ativation urves 7 14 d post-stroke: average plot of normalized I h amplitude as a funtion of memrane potential, est fitted with a Boltzmann funtion (R 2 =.99, oth fits). (d) V 5% of I h was shifted toward depolarized values after injury. (e) I h de-ativation protool. Voltage-dependene of I h de-ativation was examined y fully ativating I h at 125 and then stepping to memrane potentials etween 15 and 6. De-ativation time otant (τ D ) was alulated from exponential fits performed on the traes indiated y doule-ended arrows. (f) Expanded de-ativation urrents traes otained at 95 voltage step from the ells depited in e. (g) Time otants of I h ativation and de-ativation from the two representative ells (left) and averaged aross all the ells (right). (h) I h ativation urrents during 13 voltage step (from ells in a) were est fitted with a single-exponential funtion (gray lines). Data in,, d and g are from five ontrol and three injured rats. Data in a, e, f and g are from the same representative ontrol and injured ells. The numer of ells is indiated in the panels. Quantitative data are represented as mean ± s.e.m. *P <.5, ***P =.1,, P >.1, one-way ANOVA. advane online puliation nature NEUROSCIENCE

3 a r t i l e s 212 Nature Ameria, In. All rights reserved. Figure 3 Intra-thalami network is hyperexitale and generates epileptiform osillatio in injured animals and in a model. (a) Top, multiunit reordings in thalami slies ipsilateral to ortial stroke depiting network osillatio evoked y single eletrial shoks (arrowheads) to internal apsule. Iet, ox harts of osillation duration from three ontrol rats (n = lies) and three injured rats (n = 8 slies); one-way ANOVA. Bottom, representative spontaneous ativities. Red ox indiates the reording enlarged on the ottom trae. Note the resendo, deresendo pattern of the osillation (dots and arrows). Statistial ox harts show the mean (entral dot), median (large horizontal line), 99% and 1% range (rosses), 25% perentile range (ox), and 5 95% perentile range (whiskers). () Top, minimal thalamus model inluding a thalamoortial (TC) neuron, negative feedak through retiular thalami neuro (g GABA ) and a otant steady-state ortial input urrent (I inj ). Bottom, hanges in intrii exitaility (I h + area: altered ativation of oth I h and memrane area) promoted epileptiform thalamoortial respoe in the network. * represents the time of stimulation. () Map of osillation duration as a funtion of the retiular thalami thalamoortial feedak (g GABA ) and the injeted input urrent (I inj ) indiates that, in injured onditio, a larger hyperpolarizing urrent (I inj ) was required to prevent the initiation of osillatio. Threshold for osillation initiation refers to the threshold value of I inj aove whih an osillation is initiated. (d) Differene in threshold for osillation initiation etween ontrol and different injured onditio: θ(i h + area) (solid red) when omining altered area and altered I h ativation, θ(i h )+ θ(area) (dashed line) when adding separate effets of altered area and altered I h ativation, or following omined therapeuti onditio with modified h and leak ondutanes ( θ(t/g h + g L ), luish green). (e) Differene in osillation duration etween ontrol and injured onditio as a funtion of g GABA in the physiologial range of resting memrane potential (dashed lines). Duration was inreased in injured onditio (top) and restored to ontrol level under therapeuti onditio (ottom). I h, only I h ativation was altered (Fig. 2); area: only memrane area was altered (lower C m ; Fig. 1 and Online Methods); I h + area: oth properties were altered (Supplementary Figs. 4 and 5 for details). Cortial stroke leads to epileptiform thalami osillatio We next asked whether the inreased intrii exitaility of thalamoortial neuro tralated into altered iruit funtion in the thalamus. For this purpose, we assessed thalami network ativity in horizontal thalami slies using multiunit extraellular reordings 7 14 d poststroke. These slies oerve intra-thalami onnetivity etween the retiular thalami nuleus (nrt) and thalamoortial nulei, ut not with ortex 21. In slies from ontrol rats, eletrial stimulation of the internal apsule evoked rhythmi ativity in thalamus, oistent with previous findings (Fig. 3a) 15,22,23. The duration of the evoked ativity was inreased in slies from injured rats (Fig. 3a). In addition to enhaned evoked osillatio, post-stroke (injured) thalami slies (8 of 8 slies from 3 rats) also generated 1 5-s-long roust spontaneous epileptiform network osillatio that were never oserved in ontrol slies ( of lies from 3 rats) (Fig. 3a). This epileptiform ativity was oserved in the glioti thalamus and within ~2 µm of its order, orresponding to the order etween VPL and VPM, inluding the lateral VPM. These results indiate that ortial stroke leads, over time, to a hyperexitale intra-thalami network of surviving ells that is then ale to generate epileptiform ativities. Similar multiunit ursting ativity in thalamus was oserved during spontaneously ourring seizures in awake, ehaving rats post-stroke (Supplementary Movie 1). Computational modeling of intrathalami network ativity We assessed whether thalamoortial ellular modifiatio ould e respoile for enhaned osillatio in the thalami network in a omputational model that inluded thalamoortial and nrt neuro and a otant ortial input (Fig. 3). Using a single-ell model, we found that the omined modifiatio in memrane area and in I h (V 5% ) and faster ativation time otant (τ a ) ould aount for the post-stroke hyperexitaility in thalamoortial ells a slie Evoked osillatio 5 µv slie Osillation duration (s) Spontaneous osillatio slie slie P <.5 5 µv 5 µv 5 µv 5 µv (l h + area) 65.7 * d nrt g GABA I 72.4 Inj TC * θ (µa m 2 ) 1..5 Threshold shift θ(l h ) + θ(area) Output 25 5 ms θ(l h + area) θ(t/g h + g L ) g GABA (ms m 2 ) (Supplementary Fig. 3). We found that these intrii hanges in ell size and in I h ativation properties enhaned thalami network osillatio (Fig. 3, and Supplementary Fig. 4). Speifially, a hange in oth intrii properties redued the threshold for initiation of osillatio, as it inreased the magnitude of the hyperpolarizing urrent required to prevent the osillation (Fig. 3). Notaly, this effet on threshold resulted from a supra-linear interation of intrii hanges (Fig. 3d). Moreover, the osillation duration was enhaned y the hanges in intrii exitaility in thalamoortial neuro (Fig. 3e and Supplementary Fig. 5). Thus, the alteratio that we oserved in I h and in memrane area in injured thalamoortial ells (Figs. 1 and 2) are epilepti in that they make the thalamus osillate for longer duratio or in onditio in whih it would normally e silent. The enhaned osillatio in the injured onditio were very roust in that they were unaffeted y reduing nrt spike output (data not shown), whih would result from redued exitaility of nrt following foal ortial stroke 19. Finally, we found that we were ale to restore oth the duration and the threshold of osillation initiation y modifying I h and leak ondutanes, g h and g L, respetively (Fig. 3d,e and Supplementary Figs. 4 and 5). Cortial stroke indues thalamoortial epilepsy Chroni monitoring of ontrol (n = 7) and injured (n = 5) rats up to 1 months revealed that stroke resulted in spontaneous ital ativity only in injured rats (3 of 5 rats). Seizures were 1 12-s-long and ourred >1 month post-stroke, oistent with previous findings 17,18,24, and were assoiated with motor arrest (Supplementary Movies 2 and 3). The osillatory power of the ital eletroenephalogram (EEG) peaked in the 3 5-Hz and 8 1-Hz frequeny ands (Fig. 4a and Supplementary Fig. 6) and had a different spetral signature than typial asene seizures in rats, oistent with a previous study 17. I inj (µa m 2 ) e V Rest () V Rest () Osillation duration.5 Infinite Traient.5 Silene Infinite (l h + area).5 Traient 1. Silene g GABA (ms m 2 ) Change in osillation duration (l h + area) ontrol T/g h +g L ontrol g GABA (ms m 2 ) 2, 1,6 1,2 8 4 (ms) >2, 1, 1, < 2, (ms) nature NEUROSCIENCE advane online puliation

4 a r t i l e s Figure 4 Cortial stroke leads to late spontaneous epilepti ativities in ortex and thalamus. (a) EEG wavelet spetrogram from a representative ortial hannel (top trae). Vertial dashed lines indiate the oet and the end of the eletrographi seizure ativity. White traes represent simultaneous ortial EEG and thalami LFP reordings, temporally aligned with the wavelet spetrogram. () Representative 1-s-long ital and interital EEG reordings from the reordings depited in a. () Corresponding power spetra of ital a (Hz) 1 2 Cortex Thal Time (s) (orange) and interital (lak) EEG ativities from the ipsilateral ortex hannel. Dots indiate the dominant peak frequenies during ital periods (4 5 and 8 1 Hz). The depited reordings were otained 6.5 months after stroke indution from a 7.5-month-old rat. Contra. x., ontralateral ortex; Ipsi. x., ipsilateral ortex; Thal., thalami. 1 lpsi. x. Contra. x. Thal. 1 Thal. 2 Thal. 3 Thal. 4 Interital Ital.7 2 Hz 1 ( 1 3 ) 4 Power Frequeny (Hz) 212 Nature Ameria, In. All rights reserved. Post-stroke seizures had lear thalami involvement even though their spetral properties were distint from typial thalamoortial asene seizures. In addition, seizure-like osillatio usually spread to the ontralateral hemisphere, oistent with previous findings 24, and were synhronized in ortial EEG and thalami loal field potentials (LFPs) (Figs. 4a, and 5 and Supplementary Figs. 6a, and 7d). Reduing thalami output aorts seizures in ehaving rats Next, we asked whether thalamoortial ell ativity is important for seizure expression in vivo. To test whether seizures ould e terminated y seletively inhiiting thalamoortial neuro, we expressed either enphr3., whih enales memrane hyperpolarization and redution of ation potential firing when illuminated with yellow light 25, or eyfp under the Camk2a promoter, in the ventroasal Figure 5 Seletive optial inhiition of thalamoortial neuro interrupts ongoing epilepti seizures in awake, freely ehaving animals. (a) Diagram of hroni multisite optrode (CMO) implanted into somatoseory thalamus for ehaving reordings and optial stimulatio. Arrowheads indiate thalami reording sites ( 4). () Confoal image of oronal rain setion taken through the ortial lesion (red dashed line) showing enphrexpressing thalamoortial fiers terminating mainly in layer 4 (yellow arrow) from a rat killed after reordings. () Representative example of simultaneously reorded ortial EEGs and thalami LFPs efore and during 594-nm light delivery in the thalamus ipsilateral to stroke. Arrows indiate seizure oet and its interruption y light delivery in thalamus. (d) Mean spetrograms of thalami LFPs and ortial EEGs from the same rat. We delivered 594-nm light pulses in VPM at time. Shown are examples from all stimulatio (ital, n = 19; interital, n = 4) from a single reording session ~4.2 months post-stroke, 4 months post-viral delivery in thalamus. (e,f) Power quantifiation of ortial EEGs (e) and thalami LFPs (f) efore and during light delivery in thalamus (e left: ital, n = 56 events from 3 different trials; interital, n = 8 events from 2 different trials; e right: ital, n = 6 events from 2 Optial fier thalamus ipsilateral to ortial stroke. We then designed a devie ontaining multiple EEG eletrodes and a CMO 26 (Supplementary Fig. 7 and Online Methods) and implanted the CMO in ipsilateral ventroasal thalamus (Fig. 5a) to allow the seletive illumination of thalamoortial neuro while monitoring their firing. We also implanted four hroni EEG eletrodes, two aove peri-stroke ortex (within ~.5 mm of the edge of the lesion; see Fig. 5) and two in the orresponding ontralateral ortex (Supplementary Fig. 8 and Online Methods). This allowed us to monitor the effet of optial inhiition of thalamoortial neuro on simultaneously reorded thalamoortial multiunit firing, LFP, ortial EEG and ehavior in four rats. Seletive illumination of enphr3.-expressing thalamoortial neuro with 594-nm light interrupted ongoing eletrographi epilepti ativities in thalamus and ortex (Fig. 5,d), as well as the ehavioral seizure, and the rats immediately resumed normal ehavior a d e f Ipsi. x. Contra. x. r.m.s. power () r.m.s. power () VPM VPL Rat 1 Rat 2 Cortex Ital Interital.12 *** ***.6 Pre-light Ipsi. Contra. Ipsi. Contra. Thalamus ital.18 *** **.12 *** ***.6 RT 1 mm Light different trials; interital, n = 11 events from 2 different trials). Results in the left and right panels of e are from two different rats. The results shown in f and the left panel of e are from the same rat. r.m.s. power was averaged efore and during light delivery (see Online Methods). Error ars represent s.e.m., P.1; *P <.5, **P <.1, ***P <.1, paired t test or signed rank test, as appropriate. Results in f were otained using light at 1 mw. Oet r.m.s. power () r.m.s. power () Interruption Ital ** * Midline Cortex 1 mm Interital Ipsi. Contra. Ipsi. Contra. Thalamus interital Pre-light Light.8 Frequeny (Hz) Ital 1 2 Ipsi. x Contra. x Thalamus Thalamus Frequeny (Hz) Thalamus Thalamus Light 5 5 Time (s) Interital 1 2 Ipsi. x Contra. x. Thalamus Thalamus Thalamus Thalamus Light 5 5 Time (s) advane online puliation nature NEUROSCIENCE

5 a r t i l e s 212 Nature Ameria, In. All rights reserved. Figure 6 Multiunit firing of thalamoortial neuro in awake, freely ehaving animals. (a,) Histograms representing the mean multiunit firing frequeny of thalamoortial neuro (in, ) at the indiated times efore, during and after a 2-s light pulse delivery in the thalamus (594 nm, 1 mw, n = 9 repetitio within a trial). Multiunit firing was reorded simultaneously at four thalami loatio ( 4; see CMO diagram in Supplementary Fig. 7d). Corresponding raster plots are presented at the ottom of eah histogram. Data are from two different rats: one with enphr + eyfp + thalamoortial neuron (a) and one with enphr eyfp + thalamoortial neuro (). The rat in had a stroke, whereas the rat in a did not. (,d) Multiunit firing rate averaged efore, during and after light illumination of the thalamus. The numer of sweeps is indiated in eah graph. Eah graph in and d is from a different a Rat 1 enphr-eyfp rat, illustrating the reproduiility of light effets etween rats. Rat 5 orresponds to the Rat hown in Figure 5 e,f in whih thalami illumination disrupted seizures. Data are represented as mean ± s.e.m., P >.1; *P <.5, **P <.1, paired t test or signed rank test, as appropriate. Only data from hannels from whih we were ale to quantify firing are presented. Firing was not deteted in all of the thalami hannels. (Supplementary Movie 4). The finding that inhiition of thalamoortial neuro ipsilateral to stroke interrupted not only ipsilateral, ut also ontralateral seizures, indiates that seizures were generated in the hemisphere ipsilateral to stroke. Following interruption of the seizure y light, there was a resumption of normal thalami and ortial ativities (Fig. 5 f) oiniding with a swith to non-epilepti ehavior. a r.m.s. power ().1 *** *** Ipsi. Contra Light Cortex Cx* No light Cumul. Pro. Detetion + light 1..5 Ipsi r.m.s. power () 9 Light (n = 22) Cumul. Pro Time (s) No light (n = 33) 1..5 Contra. Detetion + no light Cortex Cx*.9.18 r.m.s. power () d Rat 2 eyfp Time (s) No light Light 14 7 Rat 3 24 enphr-eyfp 3. n = Rat 1 enphr-eyfp ** * ** ** n = 13 * * n = 9 n = 187 n = 15 Thus, seletively inhiiting thalamoortial output was suffiient to swith off the ongoing eletrographi and ehavioral seizure. Lower inteity light ( 5 mw) did not signifiantly affet thalami ativity (P >.5) and was not effetive in disrupting seizures (Supplementary Fig. 7a,,d). Notaly, the laser light at the inteity used to silene the seizures did not affet normal physiologial rhythms in ontrol non-injured animals (data not shown) or the normal EEG ativities etween seizures (referred to as interital) in epilepti animals (Fig. 5e,d and Supplementary Fig. 8a), and did not seem to affet normal interital ehavior suh as sleep (Supplementary Movie 5). In ontrast with enphr3.-tradued rats, light had no effet on eyfp-tradued rats, indiating that disruption of thalamoortial osillatio and seizures was a result of enphr3. ativation and not of nopeifi effets of light, suh as heating or visual ueing. We also onfirmed that 594-nm light illumination silened the firing of enphr3.-expressing thalamoortial neuro in vitro (n = 9 ells Pre Light Post Pre Light Post Rat 5 enphr-eyfp Pre Light Post 14 7 Rat 2 eyfp Pre Light Post Rat 4 eyfp Pre Light Post Frequeny (Hz) Cx Cx* Cx Thalamus Thalamus 1 Cx Cx* Cx.8 Figure 7 Online detetion and interruption of seizures via a 594-nm light illumination of thalamus in freely ehaving animals. (a) Following an automati detetion of seizure ativity, the system randomly triggered either yellow light (yellow) or sham stimulation (gray, no light). Shown is r.m.s. power of ipsi- and ontralateral EEGs in a 2-s period following seizure detetion in the presene of yellow light in the thalamus (n = 22 events) or during sham stimulation (n = 33 events). () Corresponding umulative proaility distriution of the average r.m.s. power with or without the light. () Top, representative spetrograms from a ortial EEG and a thalami LFP in the yellow light (left) or sham (right) onditio. Bottom, orresponding reordings of ortial EEGs (Cx) and thalami LFPs ( 4). The spetrograms and the eletrophysiologial reordings are temporally (vertially) aligned. Data in were otained using.5-s-long light pulses. Cx* indiates the ortial trae from whih the spetrogram was otained. Data are represented as mean ± s.e.m. ***P <.1. nature NEUROSCIENCE advane online puliation

6 a r t i l e s 212 Nature Ameria, In. All rights reserved. a d e from 4 rats; Supplementary Fig. 9) and redued the multiunit firing in the thalamus in enphr3.-tradued (n = 4 rats: 2 injured and 2 non-injured), ut not in eyfp-tradued, ehaving animals (n = 2 injured rats; Fig. 6). Finally, we engineered a method for deteting and silening seizures online in hronially implanted rats (Supplementary Fig. 1). An EEG hannel was routed to a real-time proessor that alulated the EEG line length 27 and triggered laser stimulation on rossing of a threshold. This system was apale of deteting and silening seizures within of initiation (Figs. 7 and 8, Supplementary Fig. 1 and Supplementary Movies 6 and 7). We were ale to validate this method up to nearly a year post-stroke in two injured rats with hroni implants (Figs. 7 and 8 and Supplementary Figs. 1 and 8). DISCUSSION We used temporally preise, ell type speifi optogeneti manipulatio to identify a neural sustrate that an e readily targeted Figure 8 Line-length alulation for real-time detetion of seizures. (a e) EEG reordings (lak traes) and their orresponding line length (red traes) alulated in real time using a 2-s sliding window (see Online Methods). Line-length threshold for seizure detetion was set manually at the eginning of the experiment. After upward rossing of the threshold (dashed line), the system randomly triggered either laser stimulation (yellow oxes) or no stimulation (gray oxes). Seizure ativity was interrupted y.5-s-long and 1-s-long 594-nm light pulses (1 mw) in real time. Data are from two different rats (one shown in a d and the other in e). Traes depited at the left are enlarged on the right for visiility. for post-injury seizure ontrol. In sum, we found that foal photothromoti stroke in the rat somatoseory ortex led to seondary hanges and hyperexitaility in the funtionally related thalamus. Changes in input resistane and I h in thalamoortial neuro enhaned their rhythmogenesis. A minimal omputational model suggested that suh hanges inrease thalami network exitaility and support epileptiform osillatio. To test this model, we designed a method for speifially silening thalamoortial neuro from this hyperexitale thalami region in real time. Reduing the thalamoortial output rapidly (<1s) interrupted the eletrographi and ehavioral seizure. Thus, we provide, to the est of our knowledge, the first evidene that thalamoortial neuronal ativity is required for post-stroke epilepsy given that reduing ativity of thalamoortial ells is suffiient to disrupt seizures either at their oet or after their generalization. Our oservation of effiient thalami inhiition was not expeted in light of the urrent working model, aording to whih epilepsy results mainly from maladaptive reorganization in ortex adjaent to the damaged area. Disruption of seizures at their oet with losed-loop ontrol, via a rief targeted inhiition of thalamoortial ells, as we found here, is a promising potential therapeuti approah for otherwise untreatale epilepsies, as it would not affet normal rain ativity etween seizures, as might other therapeuti approahes, suh as surgial lesio or hroni treatment with pharmaologial agents. It is important to note that the goal of our study was not to determine the exat site of seizure initiation. Irrespetive of the question of whether the seizure initiates in ortex or thalamus, we found that the thalami output was required to maintain the ortial eletrographi and ehavioral seizure. A major advantage of targeting thalamoortial maintenane of seizures is that this mehanism of a rapid disruption of seizures via a rief inhiition of thalamus would e synergisti with other treatments targeting, for example, seizure initiation, seizure spread or seondary epileptogenesis. Another key enefit of targeting thalamus is that it would not disrupt higher ognitive funtio that are dependent on intraortial proessing. The I h alteratio that we oserved in thalamoortial ells are similar to what have een desried in other epilepsy models 2, suggesting that these alteratio ould e a mehanism that is ommon to various epilepsies. The finding of altered intrii properties (I h and R in ) in thalamoortial neuro that enhaned rhythmogenesis led us to hypothesize that inhiiting these hyperexitale thalamoortial neuro ould rapidly aort ortial seizures. Even though I h alteratio do not entirely aount for thalami hyperexitaility, it is possile that manipulating I h, either hronially or traiently, ould also lead to seizure disruption. Indeed, as suggested y our omputational model, omining a redution in g h and an inrease in g L in thalamoortial ells is expeted to e antiepilepti. However, for real-time anti-seizure ontrol, we hose a ell type speifi optogeneti approah rather than a pharmaologial manipulation of I h. The latter would not e ell speifi or provide real-time ontrol. Future tehnologies might allow targeted real-time manipulation of ion hannel mediated urrents, suh as I h, that ould at as similarly effetive and more speifi treatments of the epilepti injury. Alternatively, the road, ut temporally speifi, redution of thalamoortial exitaility that we found may prove to e the most effetive approah. In onlusion, our findings at ellular, network and ehavioral levels provide ausal support for the hypothesis that the thalamus is ritially involved in ortial injury indued epilepsy and lead to the general onept that a struture remote from, ut onneted to, primary damaged tissue via long-range projetio an e ritially advane online puliation nature NEUROSCIENCE

7 a r t i l e s 212 Nature Ameria, In. All rights reserved. involved in anormal rain ativity suh as seizures. Our results suggest previously unknown roles for long-range onnetio in maintaining normal and pathologi osillatio, and potential future therapeuti strategies for silening speifi remote strutures in ases in whih removal of the damaged ortial area is not an option or does not result in seizure ontrol. The finding that thalamus is ritial for maintaining seizure ativity suggests that targeting a spatially restrited region of the thalamus might e a more tratale therapeuti target for neural modulation than targeting the orresponding, yet more spatially extended, ortial anormality. Methods Methods and any assoiated referenes are availale in the online version of the paper. Note: Supplementary information is availale in the online version of the paper. Aknowledgments We thank C. Pisaturo for designing and fariating ustom eletronis, A. Herert and S. Jin for their help with animal husandry, and K. Graer and D. Prine for disussio related to linial aspets of post-stroke epilepsy. J.T.P. is supported y the US National Ititute of Neurologial Disorders and Stroke (grant K99NS ) and the Epilepsy Foundation. J.R.H. is supported y grants from the US National Ititute of Neurologial Disorders and Stroke (5R1NS6477 and 5R1NS34774). T.J.D. is supported y a Berry Foundation Postdotoral fellowship. K.D. is supported y the Howard Hughes Medial Ititute, the California Ititute for Regenerative Mediine, the US National Ititutes of Health and the Defene Advaned Researh Projets Ageny (DARPA) Reorganization and Plastiity to Aelerate Injury Reovery (REPAIR) Program. E.S.F. is supported y a Epilepsy Foundation Postdotoral Fellowship. AUTHOR CONTRIBUTIONS J.T.P. and J.R.H. designed the experiments and wrote the manusript. J.T.P. performed all of the in vitro experiments. J.T.P. and T.J.D. designed and performed the in vivo experiments. B.D. performed omputational modeling. K.P. performed pilot EEG reordings. I.P. performed histology. J.T.P., J.R.H. and E.S.F. analyzed data. K.D. provided reagents and tools. COMPETING FINANCIAL INTERESTS The authors delare no ompeting finanial interests. Pulished online at Reprints and permissio information is availale online at reprints/index.html. 1. Clarkson, A.N., Huang, B.S., Maisaa, S.E., Mody, I. & Carmihael, S.T. Reduing exessive GABA-mediated toni inhiition promotes funtional reovery after stroke. Nature 468, (21). 2. Kotila, M. & Waltimo, O. Epilepsy after stroke. Epilepsia 33, (1992). 3. Kelly, K.M. Animal modeling of poststroke seizures and epilepsy: 5-year update. Epilepsy Curr. 7, (27). 4. Lee, J.-C. et al. Seizures in hildhood ishemi stroke in Taiwan. Brain Dev. 31, (29). 5. Kwan, P. & Brodie, M.J. Early identifiation of refratory epilepsy. N. Engl. J. Med. 342, (2). 6. Loiseau, P. Pathologi proesses in the elderly and their assoiation with seizures. in Seizures and Epilepsy in the Elderly (eds. Rowan, A.J. & Ramsay, E.) (Butterworth-Heinemann, 1997). 7. Hoffman, S.N., Salin, P.A. & Prine, D.A. Chroni neoortial epileptogenesis in vitro. J. Neurophysiol. 71, (1994). 8. Huguenard, J.R. & Prine, D.A. Basi mehanisms of epilepti disharges in the thalamus. in The Thalamus: Experimental and Clinial Aspets (eds. Steriade, M., Jones, E.G. & MCormik, D.) (Elsevier, 1997). 9. Bruehl, C., Kloier, O., Hossman, K.A., Dorn, T. & Witte, O.W. Regional hypometaolism in an aute model of foal epilepti ativity in the rat. Eur. J. Neurosi. 7, (1995). 1. Detre, J.A., Alsop, D.C., Aguirre, G.K. & Sperling, M.R. Coupling of ortial and thalami ital ativity in human partial epilepsy: demotration y funtional magneti resonane imaging. Epilepsia 37, (1996). 11. Redeker, C., Bruehl, C., Hagemann, G., Binus, O. & Witte, O.W. Coupling of ortial and thalami metaolism in experimentally indued visual and somatoseory foal epilepsy. Epilepsy Res. 27, (1997). 12. Gasteiger, E.L., Alowitz, B. & Barken, F.M. Interital afterdisharge in foal peniillin epilepsy: lok y thalami ooling. Exp. Neurol. 88, (1985). 13. Mondragon, S. & Lamarhe, M. Suppression of motor seizures after speifi thalamotomy in hroni epilepti monkeys. Epilepsy Res. 5, (199). 14. Tye, K.M. & Deisseroth, K. Optogeneti investigation of neural iruits underlying rain disease in animal models. Nat. Rev. Neurosi. 13, (212). 15. Paz, J.T. et al. A new mode of ortiothalami tramission revealed in the Gria4 / model of asene epilepsy. Nat. Neurosi. 14, (211). 16. Tønnesen, J., Søreen, A.T., Deisseroth, K., Lunderg, C. & Kokaia, M. Optogeneti ontrol of epileptiform ativity. Pro. Natl. Aad. Si. USA 16, (29). 17. Kelly, K.M. et al. Photothromoti rain infartion results in seizure ativity in aging Fisher 344 and Sprague Dawley rats. Epilepsy Res. 47, (21). 18. Kharlamov, E.A., Jukkola, P.I., Shmitt, K.L. & Kelly, K.M. Eletroehavioral harateristis of epilepti rats following photothromoti rain infartion. Epilepsy Res. 56, (23). 19. Paz, J.T., Christian, C.A., Parada, I., Prine, D.A. & Huguenard, J.R. Foal ortial infarts alter intrii exitaility and synapti exitation in the retiular thalami nuleus. J. Neurosi. 3, (21). 2. Reid, C.A., Phillips, A.M. & Petrou, S. HCN hannelopathies: pathophysiology in geneti epilepsy and therapeuti impliatio. Br. J. Pharmaol. 165, (212). 21. Huguenard, J.R. & Prine, D.A. Intrathalami rhythmiity studied in vitro: nominal T-urrent modulation auses roust antiosillatory effets. J. Neurosi. 14, (1994). 22. Shofield, C.M., Kleiman-Weiner, M., Rudolph, U. & Huguenard, J.R. A gain in GABA A reeptor synapti strength in thalamus redues osillatory ativity and asene seizures. Pro. Natl. Aad. Si. USA 16, (29). 23. Bryant, A.S., Li, B., Beenhakker, M.P. & Huguenard, J.R. Maintenane of thalami epileptiform ativity depends on the astroyti glutamate-glutamine yle. J. Neurophysiol. 12, (29). 24. Karhunen, H. et al. Epileptogenesis after ortial photothromoti rain lesion in rats. Neurosiene 148, (27). 25. Gradinaru, V. et al. Moleular and ellular approahes for diversifying and extending optogenetis. Cell 141, (21). 26. Yizhar, O. et al. Neoortial exitation/inhiition alane in information proessing and soial dysfuntion. Nature 477, (211). 27. Esteller, R., Ehauz, J., Theng, T., Litt, B. & Pless, B. Line length: an effiient feature for seizure oet detetion. Eng. Med. Biol. So. Pro. 23rd Ann. Int. Conf. IEEE 2, (21). nature NEUROSCIENCE advane online puliation

8 212 Nature Ameria, In. All rights reserved. ONLINE METHODS We performed all of the experiments aording to protools approved y the Stanford Ititutional Animal Care and Use Committee, and every preaution was taken to minimize stress and the numer of animals used in eah series of experiments. Cortial photothromoti stroke. We performed photothromosis as desried previously 19 on Sprague Dawley rats on postnatal days 25 3 (P25 3). Briefly, we anesthetized the rats, injeted the light-seitive Rose Bengal dye (4 mg per kg of ody weight, Sigma-Aldrih) into the tail vein and foused a light from a 3-mm-diameter fier opti ale on the skull. The optial system was designed to have an emission spetrum that enompassed the in vivo asorption range of Rose Bengal (maximum asorane at 562 nm). To indue a foal photothromoti lesion in the right somatoseory ortex, we entered the light eam mm lateral and 2.5 mm audal to regma. littermate rats reeived the same injetion of Rose Bengal, ut were not photostimulated. Slie preparation. We anesthetized injured and ontrol littermate rats (P3 18) with pentoarital (1 mg per kg, intraperitoneal) and deapitated them. We prepared thalami slies as previously desried 15. Thalami osillatio. We reorded extraellular multiunit ativity in horizontal slies (4 µm) ontaining somatoseory thalamus as desried 15. Briefly, we plaed thalami slies in an interfae hamer at 34 C and superfused them at a rate of 2 ml min 1 with oxygenated artifiial ererospinal fluid ontaining 126 mm NaCl, 2.5 mm KCl, 1.25 mm NaH 2 PO 4, 2 mm MgCl 2, 2 mm CaCl 2, 26 mm NaHCO 3 and 1 mm gluose, equilirated with 95% O 2 and 5% CO 2, ph 7.4, supplemented with.3 mm glutamine 23. We performed extraellular multiunit reordings with monopolar tungsten miroeletrodes (5 1 k, FHC) plaed in ventroasal thalamus. Signals were amplified 1, times and and-pass filtered etween 1 Hz and 3 khz. We delivered eletrial stimuli to the internal apsule with a pair of tungsten miroeletrodes (5 1 kω, FHC). The stimuli were 1 µs in duration, 5 V in amplitude and delivered one every 3 s. Whole-ell path-lamp eletrophysiology from thalami slies. We performed the reordings as previously desried 15. We visually identified nrt and thalamoortial neuro using differential ontrast optis with a Zeiss (Oerkohen) Axioskop mirosope and an infrared video amera. Reording eletrodes made of orosiliate glass had a resistane of MΩ when filled with intraellular solution. During reordings, we filled the ells with.2.5% (w/v) ioytin (Sigma- Aldrih) inluded in the internal solution. We orreted the potentials for 15- liquid juntion potential. We performed the reordings in the presene of GABA A reeptor antagonist pirotoxin (5 µm, Toris). We loked GABA A reeptors to determine whether the inreased input resistane of thalamoortial neuro in injured versus ontrol rats resulted from hanges in GABA A reeptor mediated ondutane, whih we suspeted, given that the inhiitory retiular thalami thalamoortial pathway is markedly redued after stroke 19. We monitored the aess resistane in all the reordings, and inluded ells for analysis only if the aess resistane was <18 MΩ and the hange of resistane was <2% over the ourse of the experiment. We fixed whole slies and proessed them using the standard avidin iotin peroxidase method 28,29. We proessed the slies for immunofluoresene for glial firillary aidi protein (GFAP, Millipore) 19. We assessed immunofluoresene with a laser onfoal mirosope (Zeiss LSM 51). I h ativation and de-ativation urves 3. The I h ativation urve was otruted y measuring tail urrents eliited y repolarizing the memrane to 65- holding level following voltage steps etween 6 to 135 (Fig. 2a,). We measured I h at the tail urrent.7 s after the test voltage pulse (Fig. 2a) to minimize the ontriution of apaitative or non-i h ative urrents y allowing suffiient time for these events to dissipate efore measuring I h (ref. 3). We normalized the tail urrent amplitudes (I tail ) to the maximal amplitude (I tail max) and plotted them agait the memrane potential to whih the neuron was stepped during ativation of I h (Fig. 2a). The resulting data were then fitted with a Boltzmann funtion I A1 A2 = + A I ( V V )/ k 2 max 1+ e 5% where V 5% and k represent the half-maximal voltage and Boltzmann slope fator, respetively. A1 and A2 represent initial and final I/I max values, respetively. Over the entire voltage range tested, I h was well fitted (r =.99) y a single-exponential funtion, I( t) A A e t / t = + 1, where I(t) is the amplitude urrent at time t, A and A 1 are amplitude omponents, and τ is the time otant (Fig. 2f,g). Path lamp eletrophysiology data aquisition and analysis. We used Digidata 132 digitizer and pclamp9 (Moleular Devies) for data aquisition and analysis. We amplified the signals with Multilamp 7a (Moleular Devies), and sampled and filtered them at 1 khz. We alulated the amplitude of ation potentials as the potential differene etween their voltage threshold and the peak of the waveform. Experimental design and statistial analysis. Numerial values are given as mea ± s.e.m. unless stated otherwise. The sample sizes used are standard for the field. For statistial analyses, we assessed normality (Sigmastat) efore hoosing the relevant omparative test. We used nonparametri tests in ases in whih the normality test failed. We assessed statistial signifiane, as appropriate, y performing one-way ANOVA, the Mann-Whitney rank sum test or the Kolmogoroff- Smirnoff test. We performed statistial analysis with Sigma Stat 3.5 and Origin 7. (Miroal Software). The experiments reported here were not performed y linded oservers, as relevant analyses, suh as seizure detetion were performed y standardized and/or automated routines. Optogenetis. Stereotati viral injetio were arried out as previously desried 15. Briefly, we kept the rats under isoflurane anesthesia in a stereotati frame. We performed raniotomies so as to ause minimal damage to ortial tissue. We injeted virus arrying genes for fluoresent protei and enphr (raav5/camk2a-enphr-eyfp), or for fluoresent protei alone (raav5/ Camk2a-eYFP), stereotaxially in vivo into the right somatoseory ventroasal thalamus 1 2 weeks after stroke indution or Rose Bengal treatment in injured and ontrol rats, respetively. Injetion of viral DNA under Camk2a promoter results in expression only in exitatory thalami neuro 15. We infused 5 nl of the onentrated virus suspeion ( genome opies per milliliter) into ventroasal thalamus using a 1-µl syringe and 34-gauge needle. We ontrolled the injetion rate (1 nl min 1 ) y pump (World Preision Itruments). The stereotaxi oordinates of the injetio were mm posterior to Bregma, 2.8 mm lateral to the midline and mm elow the ortial surfae. For in vitro optogenetis and slie eletrophysiology, we killed the sujets 2 3 months after viral injetio orresponding to P1 14 and we made aute horizontal rain thalami slies for optial stimulatio and in vitro reordings. We prepared thalami slies and performed in vitro whole-ell reordings as desried aove. We visualized enphr-expressing ventroasal neuro with fluoresene mirosopy. We stimulated enphr-expressing neuro with yellow laser stimuli (594-nm,.7 2-mW, 2-ms to 5-s flashes; OEM Laser Systems) delivered with opti fier (BFL 37 3, Thor Las; Supplementary Fig. 1). At the end of the reordings, we fixed the slies with 4% paraformaldehyde (wt/vol) solution, then resetioned at 8 µm and low- and high-magnifiation images were otained with fluoresene (Nikon) and onfoal (Zeiss LSM 51) mirosopes, respetively. We otained oth oronal ortial and horizontal thalami slies from the same rats to onfirm the presene and loation of enphr-expressing ventroasal thalamoortial axo and terminals in the somatoseory ortex (Fig. 5). Thalamoortial neuro were reorded from ventroasal thalamus. enphr3. expression was restrited to glutamatergi thalamoortial neuro in ventroasal and was not oserved in retiular thalami GABAergi neuro (Supplementary Fig. 9). Virus spread was reproduile aross sujets. enphr urrent was similar in ventroasal ells from ontrol and injured rats and the stimulation inteity and duration required to silene enphr-expressing thalamoortial ell firing was reproduile and similar in ontrol and injured sujets, suggesting that virus expression in thalamoortial ells was omparale in ontrol and injured sujets. Chroni optrode reordings in freely ehaving sujets. We anesthetized the sujets weeks after viral injetio in ventroasal thalamus and implanted a hroni devie ontaining five EEG srews, one EMG wire to reord eletromyographi ativity and an optrode targeting the ventroasal thalamus in ontrol nature NEUROSCIENCE doi:1.138/nn.3269

9 212 Nature Ameria, In. All rights reserved. and injured rats. Optrodes 26 ontaining four tungsten depth eletrodes and a 2-µm ore (NA =.37) optial fier (Supplementary Fig. 7d) were stereotatially ierted to a depth of 5.3 mm (Fig. 5a). We delivered yellow laser light (594 nm) into the thalamus via an optial fier to inhiit enphr3.-expressing thalamoortial neuro. Arrays were designed to sample from a large thalami volume. EEG srews (2.38-mm-long #33SS, J.J. Morris) were attahed to stainless steel wires (Medwire 316 SS 7/44T). We stailized the devies on the skull with dental ement (C&B MetaBond). We onduted experiments in freely ehaving animals etween 2 weeks and 1 months after devie implantation. For reordings, we plaed the sujets in a dediated glass-walled hamer that allowed for video monitoring. We attahed a ustom-made uffering headstage amplifier to the animal to allow for reording from high-impedane depth LFP eletrodes, and attahed to a XLTek 32 Channel EEG headox via a 5-m, 12-hannel ale with inline eletrial ommutator (Plastis One). We sampled EEG reordings at 5 Hz. We refer to eletroortiographi signals reorded with skull srews as EEG reordings. To define whether the rat was epilepti or not, we used the lassifiation proposed y the International League Agait Epilepsy and the International Bureau for Epilepsy, that is, epilepsy an e oidered if there is an enduring alteration in the rain (here, the ortial lesion) and at least two seizures. Optial stimulation in freely ehaving sujets. For optial stimulation experiments, a 594-nm DPSS laser (OEM Laser Systems) was operated ontinuously at high output power for optimal staility. Output light power was redued y neutral deity filters, and stimulation was gated y a silent, low-lateny eam shutter (SRS-475, Stanford Researh Systems). The output eam was oupled to a 2-µm ore (NA =.37) fier opti ale, with an inline fier opti rotating joint (Dori Lees). This fier was passed through a onentri hannel in the eletrial ommutator efore attahing to the animal to allow for free rotation of the animal during extended experiments. Light power levels are reported for the end of the ale efore attahment to the implant. Real-time seizure detetion and disruption in freely ehaving animals. For real-time seizure disruption experiments, we routed a single ortial EEG hannel showing lear seizure ativity from the reording system to a programmale real-time digital signal proessor (RP2.1, Tuker-Davis Tehnologies). The proessor digitized the signal at 6 khz, applied a and-pass filter (1 4 Hz), alulated the line length 27 in a sliding window of, and updated at 6 khz. Line-length threshold for seizure detetion was set manually for eah animal at the eginning of the experiment. Following upward rossing of the threshold, the system randomly triggered either laser stimulation for either.5 or (y opening the eam shutter) or sham stimulation. An 11-s timeout was imposed after eah detetion event to prevent retriggering and allow for analysis of respoe to the stimulation. We analyzed data in Matla, using ustom software. We performed spetral analysis using the wavelet method with Matla (MathWorks) 22. EEG power quantifiation in freely ehaving sujets. A asis of Morlet wavelets from Hz was used, with ten wavelets per otave. Power at a given frequeny was otained using the inner produt of a Morlet wavelet with the raw signal, and then squared. To aount for logarithmi frequeny sampling, power was divided y the period for ias orretion 31. To remove the 1/f ias inherent in iologial signals, the power was normalized y the mean power at eah frequeny, resulting in spetrograms with unitless power (Figs. 5d and 7,d, and Supplementary Figs. 7a and 8a). In some ases, the power spetrum was smoothed using a.25-s oxar filter. Spetrograms were aligned on the asis of a signal orresponding to the opening of the laser shutter, and different trials were averaged together and plotted over time. For omputation of signal power aross the spetrum, the raw signal was andpass filtered from 1 5 Hz, and the r.m.s. amplitude was otained. The effet of the laser trigger on the EEG and LFP signals was quantified y omparing the r.m.s. averaged two seonds efore and after the trigger (Fig. 5e,f and Supplementary Figs. 7 and 9a,). Multiunit firing quantifiation in freely ehaving sujets. We reorded multiunit thalami depth reordings using a multiplexing headstage amplifier (M32, Triangle Biosystems) onneted to a multihannel neural reording system (Digital Cheetah, Neuralynx). Signals were andpass filtered from 6 6, Hz to detet spiking ativity. Defletio elow negative 4 µv were marked as putative spike events. We omputed the duration and amplitude y whih the signal exeeded threshold, and we exluded events with parameters more than.d. from the mean. For alulation of rates, events were ounted in 1 2-s i, divided y the in size. We disarded trials in whih there was a traient of greater than 5 µv or the ount was 3 s.d. away from the median. Bi with the same temporal offset were used to alulate average spike rates aross trials. Immunohistohemistry. We performed histology as desried previously 19. Briefly, animals that had undergone ehavioral analysis were anesthetized with pentoarital (2 mg per kg, intraperitoneal) and perfused traardially with saline followed y 4% paraformaldehyde (Sigma-Aldrih) in.1 M phosphate uffer (Sigma-Aldrih), ph 7.4. We removed the rai and postfixed them in 4% phosphate-uffered paraformaldehyde at 4 C overnight. We took digital images for doumentation of the position and extent of lesio (Supplementary Fig. 6) and we ryoproteted the rai with 3% (w/v) surose. We otained horizontal or oronal 5-µm setio with a sliding freezing mirotome (Mirom, HM 4). We proessed setio for immunofluoresene for GFAP (1:1,, Millipore AB584), and in some ases for HCN2 and HCN4 (1:5, kindly provided y R. Shigemoto, National Ititutes for Physiologial Sienes, Japan.). We inuated the setio for 1 h in normal donkey serum and then in primary antiodies diluted in phosphate-uffered saline with Triton X-1 for 48 h, ried them in phosphate-uffered saline and inuated them with a seondary fluoresent antiody (Jakson ImmunoResearh ) at a onentration of 1 µg ml 1. We mounted the setio on slides using Vetashield Mounting Media (Vetor Las) and assessed the immunofluoresene with a laser onfoal mirosope (Zeiss LSM 51). Morphologial identifiation and HCN2 and HCN4 protein quantifiation. We filled the ells with.2.5% ioytin (Sigma-Aldrih) inluded in the internal solution of the reording eletrode. We fixed whole slies and proessed them using the standard avidin iotin peroxidase method 28,29. We proessed slies for immunofluoresene for GFAP (Millipore), HCN2 and HCN4 (kindly provided R. Shigemoto). We assessed immunofluoresene with a laser onfoal mirosope (Zeiss LSM 51). We assessed HCN2 and HCN4 protein expression for eah ioytin-filled ell y measuring the volume of HCN2 and HCN4 partiles. We otained onfoal z stak images with an optial distane of.5 µm from the soma and dendriti proesses of ioytin laeled ells. Eah z stak image was rendered into a three-dimeional image (Voloity 2.6.1, Improvision) where signals from eah fluorophore were analyzed separately. Voxels oupied y ioytin-laeled ells, and HCN 2 and HCN4 immunoreativity were deteted with oistent fluoresene signal threshold inteities throughout the analysis. Computational modeling. We uilt a minimal omputational model of the negative feedak loop etween thalamoortial ells and retiular thalami neuro to assess the osillatory respoe of thalami slies. We used a Hodgkin-Huxley style iophysial model to desrie thalamoortial ell exitaility, with parameters fitted from whole-ell reordings in ontrol and injured onditio. In the thalamoortial ell model, we oider, the potential follows C V m = ( IL + INa + IK + IT + Ih ) + Iinj + IGABA, where the leak urrent is IL = gl ( V VL ), I Na and I K were taken from ref. 32, I T from ref. 33, and Ih = ghmh ( V Vh ), with ativation following t h ( V ) m h = m h ( V ) m h with and ( ) 1 V V t h V t t t e h1/2 kh V V e h k ( ) = + ( )/ h ( ) + ( 1/ 2 )/ min max min ( ) 1 mh ( V ) = 1+ exp ( ( V Vh1 / 2 )/ kh ), with parameters fitted from whole-ell path lamp reordings. Parameters were C m = 1 µf m 2, g L =.25 ms m 2, V L = 75, g Na = 35 ms m 2, V Na = 55, s Na = 5, g K = 25 ms m 2, V K = 8, s K = 18, g T =.25 ms m 2, V T = 12, g h =.5 ms m 2, V h = 4, V h1/2 = 15, k h = 1, τ min = 1, ms and τ max = 6, ms. In injured thalamoortial neuro, we set V h1/2 = 95, τ min = 5 ms and τ max = 3,5 ms to fit the +1 shift of the steady-state ativation urve and the dereased doi:1.138/nn.3269 nature NEUROSCIENCE