TCVPL

VPM

b

a

c

0

Camk2 :eNpHRinjection

in thalamus

�

in vivoCorticalstroke

Death of CTcells

and axons

TimeDeath of TCcells

and axons

Chronic EEG/optrodeimplants for behavingrecordings/optical stim.

Awake behaving recordingsand optical stim.

10 months

Cortex

ThalamusRT

ThalamusVB

Epilepsy

TC

CT

TC

StrokePeri-stroke

Post-stroke cell death Post-stroke epilepsy/hyperexcitabilityin the surviving TC circuits

Thalamichyperexcitability

Selective optical inhibition of TC neuronsinterrupts seizures

VPL

VPM

CT

Real-time digital signal processor(calculates line-length)

EEG

Seizureonset

Line-length

Laser(594 nm)

23

EEG

Thr.

Seizureinterruption

CT

Stroke

CT

Peri-stroke

<1 week post-stroke>1 week post-stroke

EEG recording system1

Line-length

EEG

Thr.

4

Line-length

EEG

Thr.

3’

1 week 3 weeks 4 weeks

Processor detected seizurebut did not trigger light

Processor detected seizureand triggered light

RT TC

TC

Camk2 :eNpHR:eYFP�

TCVPL

VPM

CT

Stroke

CT

Peri-stroke

TC

594nm

SUPPLEMENTARY MATERIAL

Closed-loop optogenetic control of thalamus as a new toolto interrupt seizures after cortical injury

Jeanne T. Paz, Thomas J. Davidson, Eric S. Frechette, Bruno Delord, Isabel Parada,Kathy Peng, Karl Deisseroth & John R. Huguenard

Supplemental Figure 1. Experimental design.

a, Timeline showing sequence of events. Green and yellow boxes indicate experiments involving optogenetics. Light grey

box indicates time of in vitro recordings (2 days –6 months post-stroke). b, Diagrams of the thalamocortical loop comprised

of cerebral cortex, thalamocortical relay nuclei and the reticular thalamic nucleus (RT). Blue and black projections

correspond to GABAergic inhibitory and glutamatergic excitatory pathways, respectively. b, left: Cortical infarct results in

death (dashed lines) of cortical neurons and corticothalamic (CT) axons and, by the end of the first week, in death of TC

cells in VPL, and does not affect intra-RT inhibition19. VB: somatosensory ventrobasal complex. b, middle: The surviving

thalamocortical loop becomes hyperexcitable (red regions and thicker projections) and generates epilepsy. b, right:

Camk2�:eNpHR viral expression in TC neurons enables inhibition of these cells with yellow light and thus reduced

excitatory output to the cortex and interruption of seizures in awake freely behaving animals. c, Real-time detection and

interruption of seizures: 1) a cortical EEG channel recorded in the awake behaving rat was routed from the recording system

to a programmable real-time digital signal processor. 2) The processor calculated the line-length in a sliding window of 2

seconds (see Methods for details). Upon upward crossing of the line-length threshold (dashed line), the system randomly

triggered laser activation (3) or not (3’). Laser activation (3) resulted in light delivery in thalamus (4) that typically

interrupted the seizure.

InjuredControlHCN2 / Biocytin

HCN4 / Biocytin

a

HCN4/Biocytin

HCN2/Biocytin

3Volume (mm )0 50 100 150

0.0

0.5

1.0

Cum

ula

tive p

robabili

ty

ControlInjured

ControlInjured

0.0

0.5

1.0

Cum

ula

tive p

robabili

ty

3Volume (mm )0 50 100 150

Control Injured

20 mm

b

Supplemental Figure 2. Cortical stroke leads to a switch from predominant HCN2 to predominant HCN4 channels in TC neurons. a-b, HCN2 (a, Left) and HCN4 (b, Left) channel immunolabeling from representative control and injured TC neurons filled with biocytin during electrophysiological recordings from slices 7-14 days post-stroke. a, Right: Cumulative probability distributions of the volume of HCN2 particles from control TC neurons (n = 3 cells, 1200 values, 400 values per cell, from 2 rats) and injured TC neurons (n = 3 cells, 1200 values, 400 values per cell, from 2 rats) are significantly different (p<10-7 One way ANOVA Student-Newman-Keuls test). b, Right: Cumulative probability distributions of the volume of HCN4 particles from control TC neurons (n = 4 cells, 380 values, 95 values per cell, from 2 rats) and injured TC neurons (n = 4 cells, 1160 values, 290 values per cell, from 2 rats) are significantly different (p = 8.10-60, One Way ANOVA Dunn’s test). These differences in HCN subunit expression could explain at least in part the changes in biophysical properties of Ih as suggested by [32].

500 ms

25 mV

500 ms

a 25 mVb

-72.4mV -65.7mV

Supplemental Figure 3. Current-clamp membrane potential traces in a Hodgkin-Huxley model of an individual TC cell. a, Control conditions. Note the absence of action potential upon depolarizing pulses and the moderate sag, as found in whole-cell recordings. b, Injured conditions (reduced membrane area, depolarized half-activation voltage and faster activation time constant of the h conductance): Excitability is increased: action potential discharge occurs upon depolarization and the hyperpolarization-evoked sag is enlarged, consistent with the experimental observation. (a, b) Injected currents from -1.65 to 0.55 mA.cm-2. Note that the increase in hyperpolarization induced depolarizing sag (here and in figure 1a,b) results from a combined change in input resistance from cell shrinkage and from altered Ih biophysical properties and not from altered Ih expression.

c

0

400

800

1200

1600

2000

−2I (µA.cm )inj

dura

tion (

ms)

-1.0-1.5 -0.5 0 0.5

Injured

T/gh

Control

T/gLT/g +gL h

b

0

400

800

1200

1600

2000

−2I (µA.cm )inj

dura

tion (

ms)

-1.0-1.5 -0.5 0 0.5

Iharea

Control

Ih+area

Injured (area) −

20

Control

40 60 80 100−2g (mS.cm )GABA

Vm

(m

V)

−85

−75

−65

−55

0

Vm

(m

V)

a

−85

−75

−65

Injured (Ih) − Control

0

>500

<−500

(ms)

<−2000

0

>2000

d

20 40 60 80 100−2g (mS.cm )GABA

Vm

(m

V)

−85

−75

−65

−55

0

Vm

(m

V)

−85

−75

−65

<−2000

−1000

0

1000

>2000

(ms)

T/gh − Control

T/gL

Oscillation duration changes

− Control

Oscillation duration changes

Oscillation duration

Oscillation duration

Supplemental Figure 5. Model result: mapping thalamic network response after injury shows that membrane 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 (bottom), as a function of gGABA and the membrane potential (Vm). In both cases, the duration is globally increased in the physiological range of membrane potentials ([-75; -65]) mV, dashed lines). b, Oscillation duration profiles as a function of the input current in control and different injured conditions (gGABA=50 mS.cm-

2). Changes in Ih activation properties (depolarized half-activation voltage and faster activation time constant) strongly decrease the threshold for transient oscillation initiation; by contrast, the decrease in membrane area induces a smaller threshold shift but powerfully increases oscillation duration. Note also that the threshold is shifted in a supra-linear manner in the presence of both Ih and area changes. c, Changes in oscillation duration in single therapeutic conditions, compared to the control condition. A decrease in the h conductance (gh) is unable to restore the duration of oscillations to control values (top). By contrast, control durations are fully restored by an increase in the leak conductance (gL) (bottom). 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 gh restores the threshold but leaves enhanced oscillations duration, (ii) by contrast, a therapeutic increase in gL does not restore the threshold but strongly lowers oscillation duration, and (iii) the combined therapeutic modification of the gh and gL restores both threshold and duration of oscillations. “Threshold” (q) is input current Iinj threshold for initiation of oscillations.

*Seizure End06/18/2010 10:44:52

20 s

0.14mV

Interictal Ictal

0.5 s

0.2 mV

Frequency (Hz)0 5 10 15 20 25 30

5

4

3

2

01

IctalInterictal

Power spectrum2mV-4(x10 )

EMG

EEG

EMG

EEG 1

2

3

4

a b c

d

2 mmBregma -2.5 mm

13

241

2

3

4

1

2

3

4

Supplemental Figure 6. Simultaneous EEG and EMG recordings 6 weeks following a cortical stroke. a–b, Epileptiform activities in the EEG are associated with a behavioral arrest and a cessation of EMG activity. The box indicates a seizure (a). The inset indicates the location of EEG electrodes contra- (3,4) and ipsilateral (1,2) to the stroke (arrow) determined post-mortem from the same rat (scale, 2 mm). b, Expanded traces from ictal and interictal recordings depicted in a. c, Power spectrum of ictal and interictal EEG activities from peri-stroke EEG recording #1. Dots indicate the typical dominant peak frequencies (~4-5 Hz and ~8 Hz; see also Fig. 4c). Note that the peak frequency (4-5 Hz) is lower than typical absence seizures in rats. d, A Nissl-labeled coronal section taken through the lesion from a rat from sacrificed 6 months after stroke and from which chronic EEG and EMG recordings were obtained. 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 subcortical white matter without damaging the hippocampus.

Light (low power)Pre-light

0.18

0.12

0.06

0.00

T1 T2 T3 T4

Thalamus

nsns

ns ns

0 5

100

101

102

Light

-5

100

101

102

100

101

102

100

101

102

100

101

102

100

101

102

Ipsi cx

Contra cx

Thalamus T4

Thalamus T3

Thalamus T2

Thalamus T1

b

0.12

0.06

0.00

p=0.05

ns

Ipsi. Contra.

Cortexa c

VPM

VPL

RTT4

T3

T2

T1

1m

m

Fre

quency

(Hz)

T4

T3T2T1

1m

m

2 s

0.8mV

Contra cx

Ipsi cx

T1

T2

T3

T4

d

Time (s)

Dorsal

Medial

16

0

11

012

020

022

0

22

0

RM

Spow

er

(mV

)

RM

Spow

er

(mV

)Supplemental Figure 7. Low power (3-5 mW) 594 nm light is not sufficient to interrupt epileptic seizures

in freely behaving 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 T1-4 ipsilateral to cortical stroke. The depicted cortical and thalamic

spectrograms are aligned in time and were obtained from simultaneously recorded seizures. 0s corresponds to

onset of 3-5 mW 594 nm light delivery to thalamus. Note that the low power light has a small, though not

significant effect, on T4 and T3 electrodes (located within<0.5 mm from optical fiber; see b) but does not

modulate the deep thalamic channels (T2 and T1; ~ 1 mm from the optical fiber; see b). b, Left: Tip of CMO

implant for awake behaving optical stimulation and recordings in the thalamus. Red arrowheads indicate

thalamic recording sites (T1-4); black arrow indicates tip of optical fiber. 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 before and during 594 nm 3-5 mW light delivery

in the right somatosensory thalamus, ipsilateral to the cortical stroke. Power was averaged 2s before and 2s

during light delivery. Bars, mean ± s.e.m.. ns, p>0.5; paired t-test or signed rank test as appropriate. d,

Representative example traces of simultaneous cortical EEG and thalamic LFP before and during 594 nm light

delivery (yellow box) in the thalamus. Arrow indicates the onset of the seizure which is not interrupted by 3-5

mW light delivery in thalamus. Note that In deep thalamic channels (T1-T2) the ictal activity is more robust

(i.e. characterized by larger LFP spikes (d) and stronger signal power (a)) than in more superficial thalamic

electrodes T3-T4. Note also that ictal activities start earlier in T1-T2 compared with T3-T4. These findings are

in agreement with the observation that the most hyperexcitable area is between VPL and VPM (also see Fig.1).

Results in a-d and Fig. 5c,d,e left, f were obtained from the same rat. RT, VPL and VPM 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 because it

does not affect the particularly “active” thalamic channels (T1-T2; located far (~1 mm) from optical fiber)

which show the highest signal power in agreement with the presence of a more robust hyperexcitability in this

deep thalamic region close to VPL. In contrast, the higher light power (8-10 mW; see Fig. 5) interrupts seizures

presumably because it modulated all thalamic channels (T1-T4).

aF

requency

(Hz)

100

101

102

100

101

102

100

101

102

0 5-5 0 5-5Time (s)

bi

1 sLine-length

Ictal 1 Interictal

Light

Ipsi cx

Contra cx

Contra cx

Ipsi cx

Contra cx

Contra cx

Ipsi cx

Contra cx

Contra cx

Ictal 2

EEG

0.005

0.5mV

100

101

102

0 5-5

Thalamus T4

Thalamus T2

100

101

102

Time (s) Time (s)

Thalamus T4

Thalamus T2

LightLight

Thalamus T4

Light

Before light

During light

bii

0.5mV

Thalamus T2

Stroke

EEGianterior

EEGiposterior

EEGcanterior

EEGcposterior

CMOc

2 mm2 mm

Supplemental Figure 8. Thalamic illumination disrupts seizures in a freely behaving 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. 594 nm light pulses were delivered to

thalamus at time 0. The depicted spectrograms are aligned in time vertically and were obtained from

simultaneously recorded cortical and thalamic channels. Shown are examples from stimulations (ictal 1: n=5;

ictal 2: n=1; interictal: n=11) from a 2.5 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 2 spectrograms). Light had no effect on interictal EEG

activity. bi, Top: Ipsilateral cortical EEG recording. Bottom: the corresponding line-length. Upon crossing of

the line-length threshold (dashed line) the seizure onset (red box) is detected in real-time triggering a 594 nm

laser delivering light to thalamus which interrupts the seizure activity (see also Supplemental Fig. 1c). bii: 200

ms–long EEG recordings from bi 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 obtained during a

period of 1 year. Location of CMO (see Supplemental Fig. 7b) and EEG electrodes is indicated (EEGi and

EEGc: ipsi- and contralateral EEGs, respectively). Note that cerebral cortex was not damaged by chronically

implanted device for ~1 year. a-c panels and Fig. 5e right are from the same rat.

VPL

icRT

VPM

b

VPMic RT

150

100

50

0

0 5 10Light power (mW)

Peak

I (

pA

)N

pH

R

50 pA

a c

d

0

120

240

Pe

ak I

(

pA

)N

pH

R

n=9 cells

GFAP NpHR/eYFP Biocytin

GFAP NpHR/eYFP Biocytin

-70 mV

-70 mV

0.5 s

20mV

20 mm

e f

Recordingelectrode

Optical fiber

Supplemental Figure 9. Functional properties of eNpHR in TC neurons in vitro. a, Representative confocal image of a horizontal thalamic slice 3.5 months post-stroke and ~3 months after eNpHR:Camk2a construct injection in vivo in VPL and VPM thalamic nuclei. The image was taken following fixation after electrophysiological recordings of TC cells (arrows) from the same slice and after GFAP (blue), eNpHR/EYFP (green) and biocytin (red) labeling. b, Low-power videomicroscopic image of the slice showing locations of patch-clamp electrode and optical fiber through which the 594nm light was delivered to activate eNpHR. c, eNpHR photocurrent (INpHR) activation curve from a representative TC neuron was best fitted with a monoexponential function (grey line). Inset: the corresponding averaged outward INpHR traces induced by 1s-long 594 nm light (yellow bar). Each trace corresponds to an average of 5 individual traces. d, Yellow light inhibited action potential firing induced by a +120 (top) and a +160pA (bottom) current injection in a eNpHR-expressing TC neuron. c,e: Data correspond to mean ± s.e.m. (c) and (d) are from the same VPM TC neuron indicated by the right arrow in (a). e, Quantification of the peak INpHR from 9 TC neurons from 4 rats. f, High-magnification confocal image of a representative TC neuron filled with biocytin during whole-cell recording. Overlap of eNpHR/eYFP (green) and biocytin (red) gives a yellow aspect to the cell. Inset: yellow light inhibited the firing induced by a positive current injection in this TC neuron. ic, internal capsule; RT, reticular thalamic nucleus; VPL and VPM, ventroposterolateral and ventroposteromedial relay thalamic nuclei.

Supplemental Table 1. Comparison of electrical membrane properties of injured and control TC neurons.

AP amplitude (mV)

AP duration (ms)

AP threshold (mV)

Rheobase (pA) # cells # rats

Control 70.5 ± 1.5 2.6 ± 0.1 -52.1 ± 0.7 109 ± 16 19 6

Injured 67.7 ± 2.2 2.3 ± 0.1 -52.1 ± 0.9 56 ± 9 16 5

ANOVA ns ns ns p < 0.01

Action potential (AP) properties were similar in control and injured TC neurons. Rheobase, i.e. the minimal current that needs to be injected in the cell to produce an action potential firing, was lower in injured TC cells in agreement with an increased Rin (see Fig. 1). Maximal number of APs crowning the post-inhibitory rebound low threshold spike (LTS) was similar in both groups (not shown), suggesting no robust increase in T-channel expression in TC neurons. These results were quantified 7-14 days post-stroke. All values are expressed as means ± s.e.m. ns, not significant (p>0.09).