帕金森氏症（Parkinson’s disease）為全球第二常見之神經退化性疾病，由於多巴胺神經細胞退化，多巴胺含量下降，造成患者本身除了運動症狀之外，還會出現失眠、躁鬱情緒及認知障礙等等精神症狀，對患者以及照護者的生活皆帶來極大的不便，而在先前的研究中發現，帕金森氏症患者的腦區會產生頻率介於20-40 Hz之間的異常震盪，稱之為「Beta oscillation」，當給予患者治療時，此異常震盪會被抑制，於是beta震盪與運動症狀被當作是帕金森氏症的指標。
　　目前臨床上的治療方法主要採用給患者服用左旋多巴胺（Levodopa）或是多巴胺類似物等等藥物，雖然在早期治療效果極好，能延緩症狀，然而長期服用約4-7年後，發現大部分的患者皆會出現藥效減弱以及藥效異動的現象，另外還會使患者產生不自主的肌肉運動，於是治療會改採用在STN腦區埋入電極，利用深腦刺激（DBS）方式代替左旋多巴胺緩解患者的運動症狀，然而，事實上深腦刺激也仍然會引起患者產生不自主的肌肉運動，這點雖然可以經由調整刺激強度來避免，但是就必須在強度太強會產生運動障礙，與強度太小無法有效刺激之間做抉擇。
　　一直以來許多研究都證明腸道菌會影響腦部的運作，而近年來有研究發現一種精神益生菌Lactobacillus Plantarum PS128能夠提升小鼠腦區的多巴胺含量，而在本實驗室先前的研究也發現，餵食大鼠PS128約4周後，beta震盪能量下降，於是我們想探究PS128的作用機制是否與DBS或是左旋多巴胺藥物有關，以及比較PS128本身效果與另外兩者的差別。
　　結果發現PS128約可使beta震盪能量下降約57%，效果與左旋多巴胺抑制53%和深腦刺激抑制44%的beta震盪能量都是約一半左右，而觀察此三項操作倆倆之間的關係，發現在有PS128抑制beta震盪能量之下（57%），深腦刺激與左旋多巴胺仍可繼續作用，能再抑制beta震盪能量（PS128 & DBS: 74%， PS128 & Levodopa: 69%），表示PS128或許與左旋多巴胺及深腦刺激不同機制，另外，先前已有許多文獻證明深腦刺激會引起多巴胺傳導物質的釋放，而在我們的實驗結果中，左旋多巴胺抑制beta震盪效果下（53%），深腦刺激的效果被消彌（Levodopa & DBS: 56%）。
　　本文的實驗結果，除了再次證明深腦刺激機制與左旋多巴胺機制有關，由於PS128本身性質與另外兩項操作不同，是以腸道菌生成菌像後影響腦部，作用比起左旋多巴胺化學分子及深腦刺激溫和許多，並且先給予精神益生菌不會使另兩項操作無效，暗示了精神益生菌PS128未來作為輔助藥物或是治療替代方案的可能性。

As the second common neurodegenerative disease in the world, Parkinson’s disease caused by death of dopaminergic neuron and the absence of dopamine. Patients not only have movement dysfunction but also mental disorder, both leads to enormous inconvenient on patients and caregiver’s life. In the previous studies, there is an exaggerate brain wave, which frequency is about 20-40 Hz and so-called Beta oscillation, only exist in Parkinson’s disease patients. When giving medication, the power of beta oscillation is been inhibited, hence this oscillation has been seen as the marker of Parkinson’s disease.
In the early stage of Parkinson’s disease, levodopa or dopamine analog are great useful for alleviate the syndrome. However, long term drug medication was limited of their effect reduction and motor fluctuation. Deep brain stimulation (DBS) of subthalamus nucleus (STN) has been recognized as a therapeutic modality for Parkinson’s disease (PD) and taken as an alternative option in addition to levodopa treatment. Although the application of DBS and levodopa were reported to suppress the physical impairment and the pathological beta-oscillation (20-40 Hz) in the motor cortex (M1) of PD patients, adverse events like treatment unresponsiveness and dyskinesia would emerge after long term medication.
Recently, similar disruption effect on M1 beta-oscillation was found in a hemi-parkinsonian rat model by feeding a specific strain of psychobiotic- Lactobacillus Plantarum PS128. After 4 weeks of PS128 oral administration (3x1010 CFU/day), the M1 beta-oscillation of 6-OHDA unilateral injected-midbrain lesioned-rats were significantly suppressed (57% reduction) to the level similar to DBS (44%) and levodopa (53%) treated groups. Moreover, both combination treatments of PS128+DBS (74%) and PS128+levodopa (69%) exhibited significant synergistic effects when compared with DBS+levodopa (56%) treated group. Long term rectification of M1 beta-oscillation was found only in PS128 feeding rats, whereas DBS and levodopa treatment showed instant (seconds) and temporal (hours) effect, respectively.
These findings indicated that PS128 exerted therapeutic effects through different mechanisms than DBS and levodopa in hemi-parkinsonian rats. It also suggested a synergistic therapeutic role of PS128 for PD patients treated with conventional therapies.

第一章 緒論 1
1. 帕金森氏症（Parkinson’s disease）與Beta震盪 1
2. 帕金森氏症成因 2
3. 帕金森氏症之治療 4
3.1. 左旋多巴胺（L-DOPA） 4
3.2. 深腦刺激（Deep brain stimulation，DBS） 5
3.3. 潛力治療法－精神益生菌 6
第二章 實驗目的 7
第三章 材料與方法 9
1. 實驗材料 9
1.1 實驗動物 9
1.2 實驗藥品 9
1.3 實驗儀器 10
2. 動物手術及實驗方法 11
2.1 誘導單側帕金森氏症大鼠模式 11
2.2 電極植入 12
2.3.刺激、紀錄方法及數據分析 14
2.4. 量化與統計 16
2.5. 不正常腦波的生成與刺激降低 Beta 震盪實驗方法 16
3.　切片免疫染色 17
3.1 大鼠犧牲及灌流取腦 17
3.2. 冷凍切片 17
4. 精神益生菌與深腦刺激與Levodopa實驗方法 19
4.1. 實驗設計 19
4.2. 實驗餵食 20
第四章 實驗結果 21
1.　單側誘導帕金森式模式鼠 21
1.1. 帕金森氏症動物模式的誘導與判定 21
1.2. 觀察運動皮層Beta頻帶共振訊號的強度 22
2.　DBS抑制效果 22
2.1. 深腦刺激DBS會隨刺激強度降低Beta震盪訊號能量 22
2.2. 深腦刺激抑制Beta震盪效果不會受到時間影響改變 23
3. 精神益生菌PS128實驗結果 23
3.1. 長期餵食大鼠精神益生菌可抑制beta震盪能量 23
3.2. 精神益生菌與深腦刺激 23
3.3. 精神益生菌與Levodopa 24
4. Levodopa實驗結果 25
4.1. Levodopa口服餵食可短時間內抑制beta震盪能量 25
4.2. Levodopa與深腦刺激 25
第五章 結論與討論 27
第六章 圖表 31
第七章 附加圖表 41
第八章 參考文獻 43

1 Dauer, W. & Przedborski, S. Parkinson's disease: mechanisms and models. Neuron 39, 889-909 (2003).
2 Sveinbjornsdottir, S. The clinical symptoms of Parkinson's disease. J Neurochem 139 Suppl 1, 318-324, doi:10.1111/jnc.13691 (2016).
3 Anderson, K. E. Behavioral disturbances in Parkinson's disease. Dialogues Clin Neurosci 6, 323-332 (2004).
4 Fries, P. A mechanism for cognitive dynamics: neuronal communication through neuronal coherence. Trends Cogn Sci 9, 474-480, doi:10.1016/j.tics.2005.08.011 (2005).
5 Baker, S. N. Oscillatory interactions between sensorimotor cortex and the periphery. Curr Opin Neurobiol 17, 649-655, doi:10.1016/j.conb.2008.01.007 (2007).
6 Kuhn, A. A. et al. Modulation of beta oscillations in the subthalamic area during motor imagery in Parkinson's disease. Brain 129, 695-706, doi:10.1093/brain/awh715 (2006).
7 Nambu, A., Tokuno, H. & Takada, M. Functional significance of the cortico-subthalamo-pallidal 'hyperdirect' pathway. Neurosci Res 43, 111-117 (2002).
8 Bosch, C., Mailly, P., Degos, B., Deniau, J. M. & Venance, L. Preservation of the hyperdirect pathway of basal ganglia in a rodent brain slice. Neuroscience 215, 31-41, doi:10.1016/j.neuroscience.2012.04.033 (2012).
9 Fogelson, N. et al. Different functional loops between cerebral cortex and the subthalmic area in Parkinson's disease. Cereb Cortex 16, 64-75, doi:10.1093/cercor/bhi084 (2006).
10 Hirschmann, J. et al. Differential modulation of STN-cortical and cortico-muscular coherence by movement and levodopa in Parkinson's disease. Neuroimage 68, 203-213, doi:10.1016/j.neuroimage.2012.11.036 (2013).
11 Marsden, J. F., Limousin-Dowsey, P., Ashby, P., Pollak, P. & Brown, P. Subthalamic nucleus, sensorimotor cortex and muscle interrelationships in Parkinson's disease. Brain 124, 378-388 (2001).
12 Cassidy, M. et al. Movement-related changes in synchronization in the human basal ganglia. Brain 125, 1235-1246 (2002).
13 Lalo, E. et al. Patterns of bidirectional communication between cortex and basal ganglia during movement in patients with Parkinson disease. J Neurosci 28, 3008-3016, doi:10.1523/JNEUROSCI.5295-07.2008 (2008).
14 Weinberger, M. et al. Beta oscillatory activity in the subthalamic nucleus and its relation to dopaminergic response in Parkinson's disease. J Neurophysiol 96, 3248-3256, doi:10.1152/jn.00697.2006 (2006).
15 Ray, N. J. et al. Local field potential beta activity in the subthalamic nucleus of patients with Parkinson's disease is associated with improvements in bradykinesia after dopamine and deep brain stimulation. Exp Neurol 213, 108-113, doi:10.1016/j.expneurol.2008.05.008 (2008).
16 Eusebio, A. et al. Effects of low-frequency stimulation of the subthalamic nucleus on movement in Parkinson's disease. Exp Neurol 209, 125-130, doi:10.1016/j.expneurol.2007.09.007 (2008).
17 Chen, C. C. et al. Stimulation of the subthalamic region at 20 Hz slows the development of grip force in Parkinson's disease. Exp Neurol 231, 91-96, doi:10.1016/j.expneurol.2011.05.018 (2011).
18 Brown, P. Abnormal oscillatory synchronisation in the motor system leads to impaired movement. Curr Opin Neurobiol 17, 656-664, doi:10.1016/j.conb.2007.12.001 (2007).
19 Oswal, A., Brown, P. & Litvak, V. Synchronized neural oscillations and the pathophysiology of Parkinson's disease. Curr Opin Neurol 26, 662-670, doi:10.1097/WCO.0000000000000034 (2013).
20 Stein, E. & Bar-Gad, I. beta oscillations in the cortico-basal ganglia loop during parkinsonism. Exp Neurol 245, 52-59, doi:10.1016/j.expneurol.2012.07.023 (2013).
21 Levy, R. et al. Dependence of subthalamic nucleus oscillations on movement and dopamine in Parkinson's disease. Brain 125, 1196-1209 (2002).
22 Priori, A. et al. Rhythm-specific pharmacological modulation of subthalamic activity in Parkinson's disease. Exp Neurol 189, 369-379, doi:10.1016/j.expneurol.2004.06.001 (2004).
23 Brown, P. & Williams, D. Basal ganglia local field potential activity: character and functional significance in the human. Clin Neurophysiol 116, 2510-2519, doi:10.1016/j.clinph.2005.05.009 (2005).
24 Quinn, E. J. et al. Beta oscillations in freely moving Parkinson's subjects are attenuated during deep brain stimulation. Mov Disord 30, 1750-1758, doi:10.1002/mds.26376 (2015).
25 Costa, R. M. et al. Rapid alterations in corticostriatal ensemble coordination during acute dopamine-dependent motor dysfunction. Neuron 52, 359-369, doi:10.1016/j.neuron.2006.07.030 (2006).

26 Dejean, C. et al. Power fluctuations in beta and gamma frequencies in rat globus pallidus: association with specific phases of slow oscillations and differential modulation by dopamine D1 and D2 receptors. J Neurosci 31, 6098-6107, doi:10.1523/JNEUROSCI.3311-09.2011 (2011).
27 Degos, B. et al. Neuroleptic-induced catalepsy: electrophysiological mechanisms of functional recovery induced by high-frequency stimulation of the subthalamic nucleus. J Neurosci 25, 7687-7696, doi:10.1523/JNEUROSCI.1056-05.2005 (2005).
28 Mallet, N. et al. Disrupted dopamine transmission and the emergence of exaggerated beta oscillations in subthalamic nucleus and cerebral cortex. J Neurosci 28, 4795-4806, doi:10.1523/JNEUROSCI.0123-08.2008 (2008).
29 Rubino, D., Robbins, K. A. & Hatsopoulos, N. G. Propagating waves mediate information transfer in the motor cortex. Nat Neurosci 9, 1549-1557, doi:10.1038/nn1802 (2006).
30 Brazhnik, E., Novikov, N., McCoy, A. J., Cruz, A. V. & Walters, J. R. Functional correlates of exaggerated oscillatory activity in basal ganglia output in hemiparkinsonian rats. Exp Neurol 261, 563-577, doi:10.1016/j.expneurol.2014.07.010 (2014).
31 Delaville, C., McCoy, A. J., Gerber, C. M., Cruz, A. V. & Walters, J. R. Subthalamic nucleus activity in the awake hemiparkinsonian rat: relationships with motor and cognitive networks. J Neurosci 35, 6918-6930, doi:10.1523/JNEUROSCI.0587-15.2015 (2015).
32 Kim, S. W., Jang, Y. J., Chang, J. W. & Hwang, O. Degeneration of the nigrostriatal pathway and induction of motor deficit by tetrahydrobiopterin: an in vivo model relevant to Parkinson's disease. Neurobiol Dis 13, 167-176 (2003).
33 Jankovic, J. Parkinson's disease: clinical features and diagnosis. J Neurol Neurosurg Psychiatry 79, 368-376, doi:10.1136/jnnp.2007.131045 (2008).
34 Groenewegen, H. J. The basal ganglia and motor control. Neural Plast 10, 107-120, doi:10.1155/NP.2003.107 (2003).
35 Hamani, C., Saint-Cyr, J. A., Fraser, J., Kaplitt, M. & Lozano, A. M. The subthalamic nucleus in the context of movement disorders. Brain 127, 4-20, doi:10.1093/brain/awh029 (2004).
36 Qiu, M. H., Chen, M. C., Huang, Z. L. & Lu, J. Neuronal activity (c-Fos) delineating interactions of the cerebral cortex and basal ganglia. Front Neuroanat 8, 13, doi:10.3389/fnana.2014.00013 (2014).

37 Nambu, A., Takada, M., Inase, M. & Tokuno, H. Dual somatotopical representations in the primate subthalamic nucleus: evidence for ordered but reversed body-map transformations from the primary motor cortex and the supplementary motor area. J Neurosci 16, 2671-2683 (1996).
38 Nambu, A. et al. Excitatory cortical inputs to pallidal neurons via the subthalamic nucleus in the monkey. J Neurophysiol 84, 289-300, doi:10.1152/jn.2000.84.1.289 (2000).
39 Braak, H. et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 24, 197-211 (2003).
40 Wakabayashi, K. & Takahashi, H. Neuropathology of autonomic nervous system in Parkinson's disease. Eur Neurol 38 Suppl 2, 2-7, doi:10.1159/000113469 (1997).
41 Spillantini, M. G. et al. Alpha-synuclein in Lewy bodies. Nature 388, 839-840, doi:10.1038/42166 (1997).
42 Samii, A., Nutt, J. G. & Ransom, B. R. Parkinson's disease. Lancet 363, 1783-1793, doi:10.1016/S0140-6736(04)16305-8 (2004).
43 Olanow, C. W. & Tatton, W. G. Etiology and pathogenesis of Parkinson's disease. Annu Rev Neurosci 22, 123-144, doi:10.1146/annurev.neuro.22.1.123 (1999).
44 Jenner, P. Molecular mechanisms of L-DOPA-induced dyskinesia. Nat Rev Neurosci 9, 665-677, doi:10.1038/nrn2471 (2008).
45 Hauser, R. A., McDermott, M. P. & Messing, S. Factors associated with the development of motor fluctuations and dyskinesias in Parkinson disease. Arch Neurol 63, 1756-1760, doi:10.1001/archneur.63.12.1756 (2006).
46 Finlay, C. J., Duty, S. & Vernon, A. C. Brain morphometry and the neurobiology of levodopa-induced dyskinesias: current knowledge and future potential for translational pre-clinical neuroimaging studies. Front Neurol 5, 95, doi:10.3389/fneur.2014.00095 (2014).
47 Benabid, A. L., Chabardes, S., Mitrofanis, J. & Pollak, P. Deep brain stimulation of the subthalamic nucleus for the treatment of Parkinson's disease. Lancet Neurol 8, 67-81, doi:10.1016/S1474-4422(08)70291-6 (2009).
48 Aziz, T. Z., Peggs, D., Sambrook, M. A. & Crossman, A. R. Lesion of the subthalamic nucleus for the alleviation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in the primate. Mov Disord 6, 288-292, doi:10.1002/mds.870060404 (1991).


49 Benazzouz, A., Gross, C., Feger, J., Boraud, T. & Bioulac, B. Reversal of rigidity and improvement in motor performance by subthalamic high-frequency stimulation in MPTP-treated monkeys. Eur J Neurosci 5, 382-389 (1993).
50 Limousin, P. et al. Electrical stimulation of the subthalamic nucleus in advanced Parkinson's disease. N Engl J Med 339, 1105-1111, doi:10.1056/NEJM199810153391603 (1998).
51 Breit, S., Schulz, J. B. & Benabid, A. L. Deep brain stimulation. Cell Tissue Res 318, 275-288, doi:10.1007/s00441-004-0936-0 (2004).
52 Giannicola, G. et al. The effects of levodopa and ongoing deep brain stimulation on subthalamic beta oscillations in Parkinson's disease. Exp Neurol 226, 120-127, doi:10.1016/j.expneurol.2010.08.011 (2010).
53 Nimura, T. et al. Attenuation of fluctuating striatal synaptic dopamine levels in patients with Parkinson disease in response to subthalamic nucleus stimulation: a positron emission tomography study. J Neurosurg 103, 968-973, doi:10.3171/jns.2005.103.6.0968 (2005).
54 Shon, Y. M. et al. High frequency stimulation of the subthalamic nucleus evokes striatal dopamine release in a large animal model of human DBS neurosurgery. Neurosci Lett 475, 136-140, doi:10.1016/j.neulet.2010.03.060 (2010).
55 Min, H. K. et al. Dopamine Release in the Nonhuman Primate Caudate and Putamen Depends upon Site of Stimulation in the Subthalamic Nucleus. J Neurosci 36, 6022-6029, doi:10.1523/JNEUROSCI.0403-16.2016 (2016).
56 Shehab, S., D'Souza, C., Ljubisavljevic, M. & Redgrave, P. High-frequency electrical stimulation of the subthalamic nucleus excites target structures in a model using c-fos immunohistochemistry. Neuroscience 270, 212-225, doi:10.1016/j.neuroscience.2014.04.016 (2014).
57 Chuang, C. F. et al. High-Frequency Stimulation of the Subthalamic Nucleus Activates Motor Cortex Pyramidal Tract Neurons by a Process Involving Local Glutamate, GABA and Dopamine Receptors in Hemi-Parkinsonian Rats. Chin J Physiol 61, 92-105, doi:10.4077/CJP.2018.BAG561 (2018).
58 Dinan, T. G., Stanton, C. & Cryan, J. F. Psychobiotics: a novel class of psychotropic. Biol Psychiatry 74, 720-726, doi:10.1016/j.biopsych.2013.05.001 (2013).
59 Oleskin, A. V. & Shenderov, B. A. Neuromodulatory effects and targets of the SCFAs and gasotransmitters produced by the human symbiotic microbiota. Microb Ecol Health Dis 27, 30971, doi:10.3402/mehd.v27.30971 (2016).
60 Sarkar, A. et al. Psychobiotics and the Manipulation of Bacteria-Gut-Brain Signals. Trends Neurosci 39, 763-781, doi:10.1016/j.tins.2016.09.002 (2016).
61 Carabotti, M., Scirocco, A., Maselli, M. A. & Severi, C. The gut-brain axis: interactions between enteric microbiota, central and enteric nervous systems. Ann Gastroenterol 28, 203-209 (2015).
62 Moloney, R. D., Desbonnet, L., Clarke, G., Dinan, T. G. & Cryan, J. F. The microbiome: stress, health and disease. Mamm Genome 25, 49-74, doi:10.1007/s00335-013-9488-5 (2014).
63 Collins, S. M., Surette, M. & Bercik, P. The interplay between the intestinal microbiota and the brain. Nat Rev Microbiol 10, 735-742, doi:10.1038/nrmicro2876 (2012).
64 Borovikova, L. V. et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405, 458-462, doi:10.1038/35013070 (2000).
65 Ghia, J. E., Blennerhassett, P., Kumar-Ondiveeran, H., Verdu, E. F. & Collins, S. M. The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model. Gastroenterology 131, 1122-1130, doi:10.1053/j.gastro.2006.08.016 (2006).
66 Bercik, P. et al. The anxiolytic effect of Bifidobacterium longum NCC3001 involves vagal pathways for gut-brain communication. Neurogastroenterol Motil 23, 1132-1139, doi:10.1111/j.1365-2982.2011.01796.x (2011).
67 Bravo, J. A. et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci U S A 108, 16050-16055, doi:10.1073/pnas.1102999108 (2011).
68 de Lartigue, G., de La Serre, C. B. & Raybould, H. E. Vagal afferent neurons in high fat diet-induced obesity; intestinal microflora, gut inflammation and cholecystokinin. Physiol Behav 105, 100-105, doi:10.1016/j.physbeh.2011.02.040 (2011).
69 Hsiao, E. Y. et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451-1463, doi:10.1016/j.cell.2013.11.024 (2013).
70 Dupre, K. B. et al. Effects of L-dopa priming on cortical high beta and high gamma oscillatory activity in a rodent model of Parkinson's disease. Neurobiol Dis 86, 1-15, doi:10.1016/j.nbd.2015.11.009 (2016).


71 Liu, Y. W. et al. Psychotropic effects of Lactobacillus plantarum PS128 in early life-stressed and naive adult mice. Brain Res 1631, 1-12, doi:10.1016/j.brainres.2015.11.018 (2016).
72 Liu, W. H. et al. Alteration of behavior and monoamine levels attributable to Lactobacillus plantarum PS128 in germ-free mice. Behav Brain Res 298, 202-209, doi:10.1016/j.bbr.2015.10.046 (2016).
73 O'Neill, B., Patel, J. C. & Rice, M. E. Characterization of Optically and Electrically Evoked Dopamine Release in Striatal Slices from Digenic Knock-in Mice with DAT-Driven Expression of Channelrhodopsin. ACS Chem Neurosci 8, 310-319, doi:10.1021/acschemneuro.6b00300 (2017).