臨床上有許多治療癲癇的方法如藥物治療、切除手術以及電刺激術，而光基因技術是近年治療癲癇的新方法，它可由光使具感光蛋白的神經元興奮或抑制。因此，光刺激可專一控制特定的神經元而不會影響週遭的神經元，且相較於電刺激副作用也較小，此外，它也有助於探索抑制癲癇的機制。先前已有許多研究探討不同頻率電刺激抑制癲癇的效果，且發現高頻刺激(High frequency stimulation, HFS)可立即抑制癲癇，而低頻刺激(Low frequency stimulation, LFS)可降低癲癇發作的頻率，但刺激頻率和癲癇波頻率間的關係仍然未知。因此，本研究目的為發展適應性癲癇狀態控制系統，探討雙頻光刺激抑制癲癇的效果。雙頻光刺激能依據僵直期(tonic phase)或陣攣期(clonic phase)的癲癇波給予對應的高頻或低頻刺激。首先以電腦數值模擬發展適應性癲癇狀態控制系統，以二階自回歸模型估測由數學癲癇模型模擬的發作間期(interictal phase)、發作期(ictal phase)的僵直期或陣攣期的腦電訊號，並計算狀態指標再選擇對應的刺激頻率作為控制訊號。在動物實驗上，則將此系統應用於光刺激治療mhChR2光基因轉殖鼠的癲癇，首先注射4-氨基吡啶(4-aminopyridine)於小鼠右側海馬誘發急性癲癇，再比較兩種雙頻光刺激抑制癲癇的抑制率和平均抑制時間，實驗結果顯示雙頻光刺激確實能終止癲癇發作，且發現雙頻光刺激中，以低頻光刺激治療僵直棘波及高頻光刺激治療陣攣棘波較能干擾癲癇的同步性使癲癇放電停止，故此雙頻光刺激為較可行的癲癇抑制方法。

Traditional epilepsy treatments included drug therapy, focal resection and electrical stimulation. The optogenetic stimulation is a new approach of treating epilepsy by means of light to stimulate or inhibit neural circuits via viral transduction of light sensitive proteins on the neurons. However, for the optogenetic stimulation, the mechanism of stimulation frequency to seizure state was still unknown in past studies. Therefore, in this study the effect of frequency on the suppression of seizures associated with tonic phase and clonic phase was investigated. First, a series of computer simulations were performed to develop an adaptive seizure state control system. Then the dual frequency stimulation (DFS) protocols were utilized to control seizures according to the states. In animal experiment, 4-aminopyridine (4-AP) was injected into the right hippocampus of mice in order to induce an acute seizure and the system was applied to detect and control seizures in six Thy1-ChR2-YFP transgenic mice. The results showed that the DFS protocol with low frequency stimulation (LFS) for tonic phase and high frequency stimulation (HFS) for clonic phase could provide more effective means of suppressing seizures than the other. As a result, it was concluded that above-mentioned protocol could disrupt the synchrony of seizure and it may provide a practical therapy for the suppression of epilepsy.
Key words: seizures, optogenetic stimulation, 4-AP, Thy1-ChR2-YFP

[1] Commission on Classification and Terminology of the International League Against Epilepsy, "Proposal for revised clinical and electroencephalographic classification of epileptic seizures," Epilepsia, 22: 489-501, 1981.
[2] Commission on Classification and Terminology of the International League Against Epilepsy, "Proposal for revised classification of epilepsies and epileptic syndromes", Epilepsia, 30: 389-399, 1989.
[3] S. Shinnar, C. O'Dell, and A. T. Berg, "Distribution of epilepsy syndromes in a cohort of children prospectively monitored from the time of their first unprovoked seizure", Epilepsia, 40: 1378-1383, 1999.
[4] H. G. Wieser, "Mesial temporal lobe epilepsy with hippocampal sclerosis. ILAE Commission report", Epilepsia, 45.6 : 695-714, 2004.
[5] C. E.Stafstrom, and D. M. Sasaki-Adams, "NMDA-induced seizures in developing rats cause long-term learning impairment and increased seizure susceptibility", Epilepsy research, 53.1 : 129-137, 2003
[6] D. M. Durand and M. Bikson, "Suppression and control of epileptiform activity by electrical stimulation: a review", Proceedings of the IEEE, 89.7: 1065-1082, 2001.
[7] P. P. De Deyn, R. D'Hooge, B. Marescau, Y. Q. Pei, "Chemical models of epilepsy with some reference to their applicability in the development of anticonvulsants", Epilepsy research, 12.2: 87-110, 1992.
[8] F. Pena, and R. Tapia, "Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: role of glutamate-and GABA-mediated neurotransmission and of ion channels", Neuroscience, 101.3: 547-561, 2000.
[9] H. J. Luhmann, V. I. Dzhala, and Y. Ben‐Ari, "Generation and propagation of 4‐AP‐induced epileptiform activity in neonatal intact limbic structures in vitro", European Journal of Neuroscience, 12.8: 2757-2768, 2000
[10] C. C. Chiang, C. C. K Lin, M.S. Ju, and D. M. Durand, "High frequency stimulation can suppress globally seizures induced by 4‐AP in the rat hippocampus: An acute in vivo study", Brain Stimulation, 6: 180‐189, 2013.
[11] B. H. Siah., "Acute suppression of seizure by theta-burst stimulation at ventral hippocampal commissure", M. S. thesis, Dept. of Mech. Eng. NCKU, 2013E
[12] K. B. Kile, N. Tian, and D. M. Durand, "Low frequency stimulation decreases seizure activity in a mutation model of epilepsy", Epilepsia, 51.9: 1745-1753, 2010.
[13] P. Rajdev, M. Ward, P. Irazoqui, "Effect of Stimulus Parameters in the Treatment of Seizures by Electrical Stimulation in the Kainate Animal Model", International Journal of Neural Systems, 21: 151‐162, 2011.
[14] T. Wyckhuys, R. Raedt, K. Vonck, W. Wadman, P. Boon, "Comparison of hippocampal deep brain stimulation with high (130Hz) and low frequency (5Hz) on afterdischarges in kindled rats", Epilepsy Research, 88: 239‐246, 2010.
[15] A. L. Velasco, F. Velasco, M. Velasco, D. Trejo, G. Castro and J. D. Carrillo-Ruiz, "Electrical stimulation of the hippocampal epileptic foci for seizure control: a double‐blind, long‐term follow‐up study", Epilepsia, 48.10: 1895‐1903, 2007.
[16] P. Boon, K.Vonck, V. De Herdt, A. Van Dycke, M. Goethals, L. Goossens, M. Van Zandijcke, Tim. De Smedt, I. Dewaele, R. Achten, W. Wadman, F. Dewaele, J. Caemaert and D. Van Roost, "Deep brain stimulation in patients with refractory temporal lobe epilepsy", Epilepsia, 48: 1551‐1560, 2007.
[17] J. F. Tellez-Zenteno, R. S. McLachlan, A. Parrent, C. S. Kubu, and S. Wiebe, "Hippocampal electrical stimulation in mesial temporal lobe epilepsy", Neurology, 66: 1490‐1494, 2006.
[18] M. Kokaiaand M. Ledri, "An optogenetic approach in epilepsy", Neuropharmacology, 69: 89‐95, 2013.
[19] G. Nagel, T. Szellas, W. Huhn, S. Kateriya, N. Adeishvili, P. Berthold, D. Ollig, P. Hegemann, and E. Bamberg, "Channelrhodopsin‐2, a directly light‐gated cation‐selective membrane channel", Proceedings of the National Academy of Sciences, 100: 13940-13945, 2003.
[20] B. Schobert and J. K. Lanyi, "Halorhodopsin is a light‐driven cholride pump", Journal of Biological Chemistry, 257: 306‐313, 1982.
[21] E. S Boyden, F. Zhang, E. Bamberg, G. Nagel and K. Deisseroth, "Millisecond‐timescale genetically targeted optical control of neural activity", Nature Neuroscience, 8: 1263‐1268, 2005
[22] E. S Boyden, "A history of optogenetics: the development of tools for controlling brain circuits with light", F1000 Biology Reports, 2011
[23] I. Diester, M. Kaufman, T., M. Mogri, R. Pashaie, W. Goo, O. Yizhar, C. Ramakrishnan, K. Deisseroth, and K. V. Shenoy, 'An optogenetic toolbox designed for primates', Nature neuroscience, 14.3: 387-397, 2011
[24] J. Tønnesen, A. T. Sørensen, K. Deisseroth, C. Lundberg, and M. Kokaia, 'Optogenetic control of epileptiform activity', Proceedings of the National Academy of Sciences, 106.29: 12162-12167, 2009
[25] J. Wang, D. A. Borton, J. Zhang, R. D. Burwell, and A. V. Nurmikko, "A neurophotonic device for stimulation and recording of neural microcircuits", IEEE EMBS, 2935-2938, 2010
[26] E. Krook‐Magnuson, et al., "On‐demand optogenetic control of spontaneous
seizures in temporal lobe epilepsy", Nature Communications, 4: 1376, 2013.
[27] C.C. Chiang, T.P. Ladas, L.E. Gonzalez-Reyes and D.M. Durand, "Seizure suppression by high frequency optogenetic stimulation using in vitro and in vivo animal models of epilepsy", Brain Stimulation, 7: 890-899, 2014.
[28] W. J. Freeman, "Model of the dynamics of neural populations", Contemporary Clinical Neurophysiology, 34: 9-18, 1978.
[29] W. J. Freeman, "Simulation of chaotic EEG patterns with a dynamic model of the olfactory system", Biological Cybernetics, 56: 139-150, 1987
[30] F. H. L. d. Silva, A. Hoeks, H. Smits, and L. H. Zetterberg, "Model of brain rhythmic activity," Kybernetik, 15: 27-37, 1974.
[31] B. H. Jansen, and V. G. Rit, "Electroencephalogram and visual evoked potential generation in a mathematical model of coupled cortical columns," Biological Cybernetics, 73: 357-366, 1995
[32] F. Wendling, F. Bartolomei, J. J. Bellanger, and P. Chauvel, "Epileptic fast activity can be explained by a model of impaired GABAergic dendritic inhibition," Eurpean Journal of Neuroscience, 15: 1499-1508, 2002.
[33] F. Wendling, A. Hernandez, J. J. Bellanger, P. Chauvel and F. Bartolomei, "Interictal to ictal transition in human temporal lobe epilepsy: insight from a computational model of intracerebral EEG," Journal of Clinical Neurophysiology, 22: 343-356, 2005.
[34] S.H. Kim, C. Faloutsos, H.J. Yang. "Coercively adjusted auto regression model for forecasting in epilepsy EEG."Computational and mathematical methods in medicine, 2013.
[35] M. K. Kıymık, I. Güler, A. Dizibüyük and M. Akın. "Comparison of STFT and wavelet transform methods in determining epileptic seizure activity in EEG signals for real-time application."Computers in biology and medicine, 35.7: 603-616, 2005
[36] R. Yadav, M. N. S. Swamy, and R. Agarwal. "STFT-based segmentation in model-based seizure detection", IEEE, 729-732, 2007.
[37] M. E. Saab and J. GotmanSaab, "A system to detect the onset of epileptic seizures in scalp EEG," Clinical Neurophysiology, 116.2: 427-442, 2005
[38] B. R. Arenkiel, J. Peca, I. G. Davison, C. Feliciano, K. Deisseroth, G. J. Augustine, M. D. Ehlers and G. Feng, 'In Vivo Light-Induced Activation of Neural Circuitry in Transgenic Mice Expressing Channelrhodopsin-2', Neuron, 54: 205-218, 2007
[39] C. C. McIntyre, W. M. Grill, D. L. Sherman and N. V. Thakor. "Cellular effects of deep brain stimulation: model‐based analysis of activation and inhibition", Journal of Neurophysiology, 91: 1457‐1469, 2004.
[40] C. Beurrier, B. Bioulac, J. Audin and C. Hammond. "High‐frequency stimulation produces a transient blockade of voltage‐gated currents in subthalamic neurons", Journal of Neurophysiology, 85: 1351‐1356, 2001.
[41] H. Luna‐Munguía, A. Meneses, F. Peña‐Ortega, A. Gaona, and L. Rocha. "Effects of hippocampal high‐frequency electrical stimulation in memory formation and their association with amino acid tissuecontent and release in normal rats", Hippocampus, 509.22: 98‐105, 2012.
[42] H. Luna-Munguia, S. Orozco-Suarez, and L. Rocha. "Effects of high frequency electrical stimulation and R‐verapamil on seizure susceptibility and glutamate and GABA release in amodel of phenytoin‐resistant seizures", Neuropharmacology, 61: 807‐814, 2011.
[43] S. Toprani and D. M. Durand. "Fiber tract stimulation can reduce epileptiform
activity in an in‐vitro bilateral hippocampal slice preparation," Experimental
Neurology, 240: 28‐43, 2013.
[44] S. Toprani and D. M. Durand. "Long‐lasting hyperpolarization underlies seizure
reduction by low frequency deep brain electrical stimulation," The Journal of
Physiology, 2013.
[45] T. I. Netoff and S. J. Schiff, "Decreased neuronal synchronization during experimental seizures", The Journal of neuroscience, 22.16: 7297-7307, 2002.
[46] B. Beverlin II., and T. I. Netoff, "Dynamic control of modeled tonic-clonic seizure states with closed-loop stimulation", Frontiers in neural circuits 6, 2012.
[47] B. Beverlin II, J. Kakalios, D. Nykamp, and T. I. Netoff, "Dynamical changes in neurons during seizures determine tonic to clonic shift", Journal of computational neuroscience, 33.1: 41-51, 2011.
[48] M. H. Chase, Y. Nakamura, C. D. Clemente, and M. B. Sterman, "Afferent vagal stimulation: neurographic correlates of induced EEG synchronization and desynchronization", Brain research, 5: 236-249, 1967.
[49] J. Magnes, G. Moruzzi, and O. Pompeiano, "Synchronization of the EEG produced by low-frequency electrical stimulation of the region of the solitary tract", Archives italiennes de biologie, 99.1: 33-67, 1961.
[50] K. Jerger and S. J. Schiff, "Periodic pacing an in vitro epileptic focus", Journal of neurophysiology, 73.2: 876-879, 1995.
[51] X‐G. Li, P. Somogyi, A. Ylinen, and G. Buzsaki, "The hippocampal CA3 network: an in vivo intracellular labeling study", Journal of Comparative Neurology, 339.2: 181-208, 1994.
[52] A. Sik, M. Penttonen, A. Ylinen and G. Buzsáki, "Hippocampal CA1 interneurons: an in vivo intracellular labeling study", The Journal of neuroscience, 15.10: 6651-6665, 1995
[53] A. K. Golińska, "Coherence function in biomedical signal processing: a short review of applications in Neurology, Cardiology and Gynecology", Studies in Logic, Grammar and Rhetoric, 25.38: 73-82, 2011
[54] L. Faes, G. Nollo, and R. Antolini. "Investigating the level of significance of the coherence function in cardiovascular variability analysis", Computers in Cardiology 2001 IEEE, 2001.