SUPPLEMENTARY MATERIAL

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1 SUPPLEMENTARY MATERIAL Closed-loop optogenetic control of thalamus as a new tool to interrupt seizures after cortical injury Jeanne T. Paz, Thomas J. Davidson, Eric S. Frechette, Bruno Delord, Isael Parada, Kathy Peng, Karl Deisseroth & John R. Huguenard a Cortical stroke Camk :enphr injection in vivo in thalamus Chronic EEG/optrode implants for ehaving recordings/optical stim. Awake ehaving recordings and optical stim. 1 week weeks weeks 1 months Death of cells and axons Death of cells and axons Thalamic hyperexcitaility RT Epilepsy Time Post-stroke cell death <1 week post-stroke Post-stroke epilepsy/hyperexcitaility in the surviving circuits >1 week post-stroke Selective optical inhiition of neurons interrupts seizures Cortex Peri-stroke Stroke Peri-stroke Stroke Peri-stroke Stroke Thalamus RT Thalamus VB VPL VPL VPL 59nm c EEG recording system 1 Real-time digital signal processor (calculates line-length) Seizure onset Laser (59 nm) Camk :enphr:eyfp Seizure interruption EEG EEG Thr. Line-length EEG Thr. Line-length Processor detected seizure and triggered light EEG Line-length Processor detected seizure ut did not trigger light Supplemental Figure 1. Experimental design. a, Timeline showing sequence of events. Green and yellow oxes indicate experiments involving optogenetics. Light grey ox indicates time of in vitro recordings ( days 6 months post-stroke)., Diagrams of the thalamocortical loop comprised of cereral cortex, thalamocortical relay nuclei and the reticular thalamic nucleus (RT). Blue and lack projections correspond to GABAergic inhiitory and glutamatergic excitatory pathways, respectively., left: Cortical infarct results in death (dashed lines) of cortical neurons and corticothalamic () axons and, y the end of the first week, in death of cells in VPL, and does not affect intra-rt inhiition 19. VB: somatosensory ventroasal complex., middle: Thesurviving thalamocortical loop ecomes hyperexcitale (red regions and thicker projections) and generates epilepsy., right: Camk :enphr viral expression in neurons enales inhiition of these cells with yellow light and thus reduced excitatory output to the cortex and interruption of seizures in awake freely ehaving animals. c, Real-time detection and interruption of seizures: 1) a cortical EEG channel recorded in the awake ehaving rat was routed from the recording system to a programmale real-time digital signal processor. ) The processor calculated the line-length in a sliding window of seconds (see Methods for details). Upon upward crossing of the line-length threshold (dashed line), the system randomly triggered laser activation () or not ( ). Laser activation () resulted in light delivery in thalamus () that typically interrupted the seizure. Thr.

2 a Control HCN / Biocytin Injured HCN / Biocytin Control Injured mm Cumulative proaility Cumulative proaility HCN/Biocytin Control Injured Volume (mm ) HCN/Biocytin Control Injured Volume (mm ) Supplemental Figure. Cortical stroke leads to a switch from predominant HCN to predominant HCN channels in neurons. a-, HCN (a, Left) and HCN (, Left) channel immunolaeling from representative control and injured neurons filled with iocytin during electrophysiological recordings from slices 7-1 days post-stroke. a, Right: Cumulative proaility distriutions of the volume of HCN particles from control neurons (n = cells, 1 values, values per cell, from rats) and injured neurons (n = cells, 1 values, values per cell, from rats) are significantly different (p<1-7 One way ANOVA Student-Newman-Keuls test)., Right: Cumulative proaility distriutions of the volume of HCN particles from control neurons (n = cells, 8 values, 95 values per cell, from rats) and injured neurons (n = cells, 116 values, 9 values per cell, from rats) are significantly different (p = 8.1-6, One Way ANOVA Dunn s test). These differences in HCN suunit expression could explain at least in part the changes in iophysical properties of Ih as suggested y [].

3 a 5 5 ms 5 5 ms Supplemental Figure. Current-clamp memrane potential traces in a Hodgkin-Huxley model of an individual cell. a, Control conditions. Note the asence of action potential upon depolarizing pulses and the moderate sag, as found in whole-cell recordings., Injured conditions (reduced memrane area, depolarized half-activation voltage and faster activation time constant of the h conductance): Excitaility is increased: action potential discharge occurs upon depolarization and the hyperpolarization-evoked sag is enlarged, consistent with the experimental oservation. (a, ) Injected currents from to.55 ma.cm -. Note that the increase in hyperpolarization induced depolarizing sag (here and in figure 1a,) results from a comined change in input resistance from cell shrinkage and from altered Ih iophysical properties and not from altered Ih expression.

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5 () V m () avm c V m () V m () Oscillation duration changes Injured (I h ) Control Injured (area) Control g (ms.cm GABA ) Oscillation duration changes T/g h Control T/g L Control g (ms.cm GABA ) >5 < 5 > < (ms) > 1 1 < (ms) duration (ms) d duration (ms) I (µa.cm inj ) 8 Oscillation duration Control I h area I h +area Oscillation duration Control Injured T/g h T/g L T/g L+gh I (µa.cm inj ) Supplemental Figure 5. Model result: mapping thalamic network response after injury shows that memrane area and leak conductance play predominant roles in determining the oscillation duration. a, Changes in oscillation duration for transient oscillations following modifications in Ih activation (top) and in area (ottom), as a function of g GABA and the memrane potential (Vm). In oth cases, the duration is gloally increased in the physiological range of memrane potentials ([-75; -65]), dashed lines)., Oscillation duration profiles as a function of the input current in control and different injured conditions (g GABA =5 ms.cm - ). Changes in Ih activation properties (depolarized half-activation voltage and faster activation time constant) strongly decrease the threshold for transient oscillation initiation; y contrast, the decrease in memrane area induces a smaller threshold shift ut powerfully increases oscillation duration. Note also that the threshold is shifted in a supra-linear manner in the presence of oth Ih and area changes. c, Changes in oscillation duration in single therapeutic conditions, compared to the control condition. A decrease in the h conductance (g h ) is unale to restore the duration of oscillations to control values (top). By contrast, control durations are fully restored y an increase in the leak conductance (g L ) (ottom). d, Oscillation duration profiles as a function of the input current in the control, injured and the different therapeutic conditions. The profiles illustrate how (i) a therapeutic decrease of the g h restores the threshold ut leaves enhanced oscillations duration, (ii) y contrast, a therapeutic increase in g L does not restore the threshold ut strongly lowers oscillation duration, and (iii) the comined therapeutic modification of the gh and gl restores oth threshold and duration of oscillations. Threshold (q) is input current I inj threshold for initiation of oscillations.

6 a EEG /18/1 1::5 *Seizure End EMG Interictal Ictal s Power spectrum (x1 ) 5 Ictal Interictal Frequency (Hz) - EEG1 1 EMG d c.5 s mm Bregma -.5 mm Supplemental Figure 6. Simultaneous EEG and EMG recordings 6 weeks following a cortical stroke. a, Epileptiform activities in the EEG are associated with a ehavioral arrest and a cessation of EMG activity. The ox indicates a seizure (a). The inset indicates the location of EEG electrodes contra- (,) and ipsilateral (1,) to the stroke (arrow) determined post-mortem from the same rat (scale, mm)., Expanded traces from ictal and interictal recordings depicted in a. c, Power spectrum of ictal and interictal EEG activities from peristroke EEG recording #1. Dots indicate the typical dominant peak frequencies (~-5 Hz and ~8 Hz; see also Fig. c). Note that the peak frequency (-5 Hz) is lower than typical asence seizures in rats. d, A Nissl-laeled coronal section taken through the lesion from a rat from sacrificed 6 months after stroke and from which chronic EEG and EMG recordings were otained. The stroke appears as a scarred area of cortex (dashed line: necrotic core). The stroke core was usually dislodged during tissue sectioning. Note that the lesion extends to the sucortical white matter without damaging the hippocampus.

7 a Frequency (Hz) 1 Ipsi cx Thalamus T Thalamus T T T T T1 d Ipsi cx 1mm Dorsal Medial T T T T1 VPL RT 1mm c RMS power () Thalamus T1 T T T ns ns ns ns Pre-light RMS power ().1.6. Light (low power) Cortex Ipsi. Contra. p=.5 ns 1 1 Thalamus T T Thalamus T1 1 1 T T 1-5 Light 5 Time (s) T1 s.8 Supplemental Figure 7. Low power (-5 mw) 59 nm light is not sufficient to interrupt epileptic seizures in freely ehaving animals: compare to Fig. 5 c-f. a, Averaged wavelet spectrograms from 7 seizures from one rat of cortical (ipsi- and contra-lateral to stroke) and thalamic recordings from channels T1- ipsilateral to cortical stroke. The depicted cortical and thalamic spectrograms are aligned in time and were otained from simultaneously recorded seizures. s corresponds to onset of -5 mw 59 nm light delivery to thalamus. Note that the low power light has a small, though not significant effect, on T and T electrodes (located within<.5 mm from optical fier; see ) ut does not modulate the deep thalamic channels (T and T1; ~ 1 mm from the optical fier; see )., Left: Tip of CMO implant for awake ehaving optical stimulation and recordings in the thalamus. Red arrowheads indicate thalamic recording sites (T1-); lack arrow indicates tip of optical fier. Right: Schematic diagram of the somatosensory thalamus showing location of the CMO. c, Power quantification of cortical EEGs (ipsi and contralateral to stroke) and thalamic LFPs ipsilateral to stroke efore and during 59 nm -5 mw light delivery in the right somatosensory thalamus, ipsilateral to the cortical stroke. Power was averaged s efore and s during light delivery. Bars, mean ± s.e.m.. ns, p>.5; paired t-test or signed rank test as appropriate. d, Representative example traces of simultaneous cortical EEG and thalamic LFP efore and during 59 nm light delivery (yellow ox) in the thalamus. Arrow indicates the onset of the seizure which is not interrupted y -5 mw light delivery in thalamus. Note that In deep thalamic channels (T1-T) the ictal activity is more roust (i.e. characterized y larger LFP spikes (d) and stronger signal power (a)) than in more superficial thalamic electrodes T-T. Note also that ictal activities start earlier in T1-T compared with T-T. These findings are in agreement with the oservation that the most hyperexcitale area is etween VPL and (also see Fig.1). Results in a-d and Fig. 5c,d,e left, f were otained from the same rat. RT, VPL and correspond to reticular thalamic, ventroposterolateral and ventroposteromedial thalamic nuclei, respectively. a,d are from the same trial as Fig. 5c,d. These results suggest that low power light does not efficiently disrupt seizures ecause it does not affect the particularly active thalamic channels (T1-T; located far (~1 mm) from optical fier) which show the highest signal power in agreement with the presence of a more roust hyperexcitaility in this deep thalamic region close to VPL. In contrast, the higher light power (8-1 mw; see Fig. 5) interrupts seizures presumaly ecause it modulated all thalamic channels (T1-T).

8 a 1 Ipsi cx Ictal 1 Ipsi cx Ictal Ipsi cx Interictal i EEG Line-length 1s ii Before light Frequency (Hz) Thalamus T 1 1 Thalamus T Light Thalamus T c EEGc anterior During light CMO.5 EEGi anterior 1 1 Thalamus T Thalamus T Thalamus T EEGc posterior Stroke EEGi posterior 1 1 mm 1-5 Light 5 Time (s) Light Light Time (s) Time (s) Supplemental Figure 8. Thalamic illumination disrupts seizures in a freely ehaving rat. a, Averaged wavelet spectrograms from the cortical EEGs ipsi- and contra-lateral to the stroke and from thalamic LFPs ipsilateral to stroke during ictal and interictal periods. 59 nm light pulses were delivered to thalamus at time. The depicted spectrograms are aligned in time vertically and were otained from simultaneously recorded cortical and thalamic channels. Shown are examples from stimulations (ictal 1: n=5; ictal : n=1; interictal: n=11) from a.5 month old rat; 1.5 months post-stroke and post-viral delivery in thalamus. Light disrupted seizure activities when presented either late, >5s after seizure onset (Ictal 1 spectrograms) or early, <1s after seizure onset (Ictal spectrograms). Light had no effect on interictal EEG activity. i, Top: Ipsilateral cortical EEG recording. Bottom: the corresponding line-length. Upon crossing of the line-length threshold (dashed line) the seizure onset (red ox) is detected in real-time triggering a 59 nm laser delivering light to thalamus which interrupts the seizure activity (see also Supplemental Fig. 1c). ii: ms long EEG recordings from i are enlarged. c, Brain from the same rat sacrificed and fixed for histology 1 year post-stroke, from which chronic optrode recordings/optical stimulations were regularly otained during a period of 1 year. Location of CMO (see Supplemental Fig. 7) and EEG electrodes is indicated (EEGi and EEGc: ipsi- and contralateral EEGs, respectively). Note that cereral cortex was not damaged y chronically implanted device for ~1 year. a-c panels and Fig. 5e right are from the same rat.

9 e RT VPL d Recording electrode 5 1 Light power (mw) mm -7 Optical fier ic RT f 1 5 pa GFAP NpHR/eYFP Biocytin n=9 cells Peak I NpHR (pa) ic c Peak I NpHR (pa) a GFAP -7 NpHR/eYFP Biocytin.5 s Supplemental Figure 9. Functional properties of enphr in neurons in vitro. a, Representative confocal image of a horizontal thalamic slice.5 months post-stroke and ~ months after enphr:camka construct injection in vivo in VPL and thalamic nuclei. The image was taken following fixation after electrophysiological recordings of cells (arrows) from the same slice and after GFAP (lue), enphr/eyfp (green) and iocytin (red) laeling., Low-power videomicroscopic image of the slice showing locations of patch-clamp electrode and optical fier through which the 59nm light was delivered to activate enphr. c, enphr photocurrent (INpHR) activation curve from a representative neuron was est fitted with a monoexponential function (grey line). Inset: the corresponding averaged outward INpHR traces induced y 1slong 59 nm light (yellow ar). Each trace corresponds to an average of 5 individual traces. d, Yellow light inhiited action potential firing induced y a +1 (top) and a +16pA (ottom) current injection in a enphrexpressing neuron. c,e: Data correspond to mean ± s.e.m. (c) and (d) are from the same neuron indicated y the right arrow in (a). e, Quantification of the peak INpHR from 9 neurons from rats. f, Highmagnification confocal image of a representative neuron filled with iocytin during whole-cell recording. Overlap of enphr/eyfp (green) and iocytin (red) gives a yellow aspect to the cell. Inset: yellow light inhiited the firing induced y a positive current injection in this neuron. ic, internal capsule; RT, reticular thalamic nucleus; VPL and, ventroposterolateral and ventroposteromedial relay thalamic nuclei.

10 Supplemental Tale 1. Comparison of electrical memrane properties of injured and control neurons. AP amplitude () AP duration (ms) AP threshold () Rheoase (pa) # cells # rats Control 7.5 ± ± ±.7 19 ± Injured 67.7 ±.. ± ±.9 56 ± ANOVA ns ns ns p <.1 Action potential (AP) properties were similar in control and injured neurons. Rheoase, i.e. the minimal current that needs to e injected in the cell to produce an action potential firing, was lower in injured cells in agreement with an increased Rin (see Fig. 1). Maximal numer of APs crowning the post-inhiitory reound low threshold spike (LTS) was similar in oth groups (not shown), suggesting no roust increase in T-channel expression in neurons. These results were quantified 7-1 days post-stroke. All values are expressed as means ± s.e.m. ns, not significant (p>.9).