1. Suppression of seizure-like activity by optogenetic stimulation in reticular thalamus Student: Hsueh Tai-En 薛岱恩 Department of Psychology, NTU Advisors: Dr. Shyu Bai-Chuang 徐百川 老師 Chang Wei-Jen 張瑋仁, Chang Wei-Pang 張維邦 IBMS, Academia Sinica

2. Importance of seizure Seizure is abnormal excessive or synchronous neural activity in the brain, producing a physical convulsion, minor physical signs, thought disturbances, or a combination of symptoms (Fisher et al., 2005). Seizure affects 1 % of population, and 30 % are suffered from drug-resistant epilepsy. Clinical application uses non-invasive approaches or deep brain stimulation for the treatment of drug- resistant seizures. Thalamus plays an important role on modulating the brain cortical seizure activity (Chang et al., 2011;Polack 2009; Ates et al., 2005; Blumenfeld, 2002). Wei-Pang Chang(2011). Epilepsia, 52(12)

3. Thalamocortical mechanism of seizure Jon-Paul(2003). TRENDS in Pharmacological Sciences, Vol.24, No.10 Red: inhibitory GABA-containing neuron Green: excitatory glutamate-containing neuron Reticular Thalamus Nucleus GABA

4. Relation between Seizure and reticular thalamus A gain in GABAergic inhibitory strength in thalamus reduces the length and power of absence seizure (Schofield et al., 2009). The net effect of increased GABAergic synaptic transmission within NRT reduces synchronized burst-firing of thalamic relay neurons. GABA A receptor-mediated inhibition of NRT outflow decrease the likelihood of absence seizures ( Hosford et al., 1997). GABAergic interneuron GABA Seizure

5. The problem Pharmacology Electrical stimulation  Using light stimulation

6. Optogenetic Optogenetic technology combines genetic targeting of specific neurons or proteins with optical technology for imaging or control of the targets within intact, living neural circuits (Deisseroth et al., 2006). Aravanis. J. Neural Eng,4. 2007 Pastrana. Nature Methods 8. 2011 Channelrhodopsins

7. Hypothesis: Activation of nRT will modulate cortical seizure Aim: 1. Use Pv-ChR2 mice as model, and verify the expression of channelrhodopsin. 2. Induce cortical seizure-like activity by injecting pentylenetetrazol (PTZ) (GABA A antagonist). 3. Use light to specifically activate nRT neuron output to investigate the effect of nRT activation during seizure.

8. Material & Method Pv-chr2 transgenic mice Michigan probe - In the reticular thalamic nucleus, nearly all neurons are parvalbumin-immunoreactive (Csillik et al., 2005). 16 electrode laser

9. Tungsten electrode corpus callosum (CC) stimulate Michigan probe Recording: Primary somatosensory cortex Optic fiber-optic electrode Reticular thalamus stimulate pv-chr2 transgenic mice Injected 60mg/kg PTZ Experiment setup CC S1 RT

10. Procedure Record 30 mins Light stimulate + Induce seizure Turn on light Induce seizure 6 times Light stimulation CC stimulation

11. Anti EYFPAnti PVmerge 100  m 2 mm The expression of channelrhodopsin cerebellum Reticular thalamus Olfactory bulb Hippocampus Cortex

12. Light stimulation modulates RT and thalamus firing rate Stimulation reticular thalamus(RT) Stimulation on thalamus(Th)

13. Cx Th Light stimulation in RT changes the firing pattern in cortex Primary somatosensory cortex(S1) P 1.82 mm L 2.5mm Reticular thalamus (RT) P 3.5mm L 2.0mm

14. Corpus callosum(CC) stimulation induces seizure Superficial layer 0.5 mV 300 ms Deeper layer Corpus callosum stimulate Control

15. Antiepleptic drug Ethoxusimide blocks CC induced seizure 2 mV 1 sec CC stimulation Control

16. light stimulation 5Hz pulse duration 100ms duration 20s No light stim CC stim 10HZ 500uA 4s Light stimulation suppressing CC induced seizure 2 mV 1 sec

17.  Most effective light stimulation pattern in suppressing seizure is 100Hz 30s light pulse Different light stimulation condition in suppressing seizure *

18. Light stimulation in RT is necessary for suppressing seizure >60% 30%~60% <30% Lateral 2.04mm Cortex Hippocampus Corpus callosum Reticular thalamus

19. Summary Light stimulation can activate reticular thalamus and thalamus by activating channelrhodopsin-2 on them. High frequency light is more effective in suppressing seizure. Location of optic fiber is crucial, those in the reticular thalamus(RT) and thalamus are more effective in suppressing seizure.

20. Acknowledgment Dr. Shyu Bai-Chuang N327 members: – Chang Wei-Jen – Chang Wei-Pang NPAS

21. Thanks for your attention!!

23. Mechanism of seizure suppression 130 Hz is in the range that can evoke Long-Term Potentiation (LTP) (Bliss and Collingridge, 1993;Malenka and Nicoll, 1999). It changes synapse plasticity and network activity. High synaptic activity, as is induced by HFS, would then lead to downscaling of the neuronal excitability with an increase in seizure threshold as a result. Depolarization block occlude the kindling stimulus, which may explain why HFS has more potential than LFS. Wyckhuys(2010). Epilepsy Research. 88, 239—246