文／劉姿佑　圖／成大生理所吳偉立實驗室

Microbiota regulate social behaviour via stress response neurons in the brain Wei-Li Wu et al. Nature, 595, 409–414.（2021）

「在美國訓練出厚臉皮的特質就是『問』。我問遍隔壁實驗室的管理人、技術人員、博士後、研究生，最後終於借到一整套百萬級的腦部立體定位儀研究設備的操作機會，也問到臺灣裔前輩分享獨門腦部顯微注射技術，快速學習把實驗操作上手。把握別人沒排程的空檔，經常從早上5點開始進行手術到晚上12點，靠這樣的方式才能完成許多高難度的腦部顯微注射實驗。」成大生理所助理教授吳偉立以論文第一作者與通訊作者身分，投稿國際知名期刊《Nature》前後歷時2年5個月，加上整個實驗過程共計將近６年，終於看到研究成果刊登在《Nature》。

“One thing I learned in the United States, is ‘don’t be shy to ask.’ After asking the lab manager, technicians, postdocs, and graduate students in the lab next door, I finally had the opportunity to operate a complete set of million-level brain stereotaxic instrument, which I borrowed. I also asked senior technician from Taiwan to share their unique brain microinjection technique and quickly learned how to operate the instrument. Making the best use of the time slots that are not booked, I often carry out surgeries from 5 AM in the morning till midnight. I was able to complete many challenging brain microinjection experiments this way.” As the first author and corresponding author of the paper, Assistant Professor Wei-Li Wu of the Department of Physiology, College of Medicine, National Cheng Kung University (NCKU), spent 2 years and 5 months submitting the paper, together with a series of experiments that stretched the overall process to 6 years, before submitting it to the internationally renowned journal “Nature,” where he finally saw the fruits of his endeavors published.  

吳偉立說，這個研究從實驗到發表的時間真的很久，中間挫折超級無敵多，數度非常想放棄轉投稿至其他期刊，但「還好我有堅持下來！也深刻體會到，每一個階段做好每件事情真的很重要。」

This research took a long period of time from experiment to publication. Wu had numerous setbacks and, at several moments, wanted to give up and re-submit the paper to other journals. “Fortunately, I persisted! I also had a profound realization that doing the best at every stage is really crucial.”

成大生理所吳偉立老師團隊破解腸道菌影響動物社交行為登上國際頂尖期刊《Nature》

The NCKU team, led by Professor Wei-Li Wu of the Department of Physiology, solved the mystery of how gut microbiota influence animal social behaviors. Their results were published in the top scientific journal Nature.

國立成功大學醫學院生理學科暨研究所助理教授吳偉立研究團隊發現腸道菌可透過腦部特定的神經迴路調控動物的壓力反應與社交行為，社交行為在群居動物中可提升生存、提供安全感、降低能量消耗等功能。經實驗證實，他們找到糞腸球菌透過腸道、大腦的溝通機制，抑制小鼠壓力賀爾蒙，並促進其社交行為。該研究成果2021年6月30日刊登在國際權威期刊《Nature》自然雜誌科學期刊。

The research team, led by Wei-Li Wu, discovered that gut microbiota can regulate the stress response and social behavior of animals through specific neural circuits in the brain. Social behavior can increase survival, provide a sense of security, and reduce energy expenditure in social animals. The team’s experiments verified their discovery that the Enterococcus faecalis inhibits stress hormone in mice and promotes their social behavior through the bidirectional communication mechanism between the intestine and brain. The findings were published in the prestigious scientific journal Nature on June 30th, 2021.

「腸其實是一個相當複雜的器官，寄居著上千種不同細菌，數量高達上百兆，有多種可能性可以影響實驗結果」。在吳偉立的研究文章中提出洋洋灑灑至少167張的數據圖，是多項扎實的實驗逐漸累積、堆疊的結果，因此，吳偉立也鼓勵研究生，「如果單單想到生命科學還存在那麼多未解決的問題，想一想真的是很頭痛，但是也不需要自己嚇自己，抱持好奇心持續探索，終究會讓事物的道理連接起來。」

“The intestine is actually a very complex organ inhabited by thousands of different bacteria, the number of which is as high as the trillions, leading to countless possibilities that can affect experimental results.” Wu’s article contained at least 167 data panels, which were the result of the accumulation and stacking of multiple robust experiments. Wu encouraged graduate students. “It’s really a headache to think that there are still many unsolved questions in biological sciences, but you don’t need to be intimated by this fact. Keep exploring with curiosity, and you will eventually connect the dots to find the truth. That’s exactly what I did for this paper.”

吳偉立表示，生理學涉及很多複雜的身體系統交互作用，仍然有許多未知仍須探索，實驗過程有時候難以預測結果，結果總是存在三種情況，數據是組別間沒有差異、實驗組增高、或降低，每一個結果應該會為我們帶來不同的推測，也才能繼續的去設計下一個實驗，「生物學很少有絕對的事情存在，當下堅持把每件事情做好，最後就能像拼圖一樣，拼起來，說出一個道理」。

Wu remarked that physiology involves many complex body system interactions, and there are still many unknowns that require exploration. Only three possibilities will show up in the experiments: no difference between the groups, or an increase or decrease in the experimental group. The result, which usually falls into one of the three situations above, are sometimes difficult to predict during the experiment. Each result should lead us to different predictions, according to which we can design the subsequent experiment. “There are very few absolute things in biology. If you insist on perfecting everything at the moment, you can finally put them together like a jigsaw puzzle and discover the truth.”

「我常鼓勵與提醒學生，回頭檢視已經完成的實驗有無什麼狀況發生」，吳偉立舉例，如果因為不明原因生病的老鼠牠們的行為表現當然會影響實驗結果，但這並不是我們要的。此外觀察動物行為時，常常分析行為30分鐘的總和不一定有差異，但把30分鐘細分為6個5分鐘來進行分析，仔細的回顧每個環節或許就會看見差異性。

“I often encourage and remind students to look back at completed experiments to see if there’s anything happening.” Wu provided an example: if the result comes from the mice falling sick for unknown reasons and exhibiting different behavior patterns, it is not the result that they want. Moreover, when observing animal behavior, for example, the overall 30-minute analysis of behavior does not necessarily yield different results, but plotting the analysis into 6 sessions of 5-minute analyses and carefully reviewing each may reveal the difference.

2021年7月14日上午，科技部在臺北舉辦線上成果發表會。科技部表示，該研究成果解開了腸道中寄居的細菌如何影響宿主社交行為之謎，而社交行為的缺損是自閉症、思覺失調症、憂鬱症、社交焦慮症等失調症的症狀之一。此研究成果受到國內外重視，也是將臺灣基礎科學研究實力推向國際的最佳典範。科技部生科司司長陳鴻震表示，吳偉立的研究是重要科學突破，備受國內外矚目。吳偉立是臺灣培育的年輕學者，帶領臺灣學生主導重要議題，並在國際頂尖期刊發表成果，為國內科學研究樹立典範。

On the morning of July 14th, 2021, the Ministry of Science and Technology (MOST) held an online conference in Taipei. According to MOST, the research results have solved the mystery of how bacteria living in the intestine affect the host’s social behavior, the deficits of which are one of the symptoms of disorders such as autism, schizophrenia, depression, and social anxiety disorder. The results promote Taiwan’s strength in basic scientific research to the global scientific community. Dr. Hong-Chen Chen, Director General of MOST’s Department of Life Sciences, said that Wu’s research is an important scientific breakthrough that has attracted attention in Taiwan and abroad. A younger-generation scholar cultivated in Taiwan, Wu has led Taiwanese students exploring important areas and publishing their results in top international journals, setting an example for domestic scientific research.

成大校長蘇慧貞表示，堅持跟自律，沒有一段路是浪費的。吳偉立為了登上《Nature》堅持多年，成大能共同成就新進教師專注研究，是老中青三代學者共同開創的臺灣新科學研究態度。

Dr. Huey-Jen Jenny Su, President of NCKU, said persistence and self-discipline make no journey wasted. Wu persisted in his research for many years to have his work published in Nature. The joint achievement in the dedicated research by newly recruited teachers in NCKU is the result of a new scientific research attitude initiated by three generations of scholars across different age groups.   

成大生理所助理教授吳偉立認為做研究在每一個階段做好每件事情很重要

Assistant Professor Wei-Li Wu of NCKU’s Department of Physiology believes that it is important to perfect every element at each stage during research

吳偉立大學主修海洋生物學，接受了很多生態、分類、演化的理論薰陶；碩士論文研究蓋斑鬥魚打架時腦部神經傳導與身體表徵體色的關係；博士論文的研究是探討周邊神經系統如何影響中樞神經所調控的行為表現；博士後研究最初的研究方向則跟母體免疫反應造成胎兒神經發育與子代行為發展有關；博士後研究後半段則是研究腸道共生細菌透過腸腦軸調控神經與行為表現。

Wu majored in marine biology and was nurtured by various ecological, taxonomic classification, and evolutionary theories. His master’s thesis focused on the relationship between neurotransmission and body colors during the fights of Macropodus opercularis (paradise fish). For his doctoral dissertation, Wu explored how the peripheral nervous system affects the behavioral output regulated by the central nervous system. Subsequently, his postdoctoral research was initially related to the effects maternal immune response has on the neural development of the fetus and the behavioral development of the offspring; the later part of his postdoctoral research focused on how gut microbiota regulate nervous and behavioral performance through the gut–brain axis.

各個階段的研究主軸看似各不相同，吳偉立認為，這些不同經驗的累積造就了今天的結果，「過去走過的看似截然不同的道路，似乎會在某個時間點連結起來，以本項研究為例，腸道微生物是超級複雜的生命體，腸道內就像一個微型的生態系統，要找出它們跟大腦之間的溝通方式以及證明是哪些種類的細菌真的參與其中，背後理論機制如果從生態學角度切入思考，會有另外一番道理存在」。

The focus of his research at each stage seemed to differ. Wu believes that his results today are an accumulation of different experience. “The seemingly different paths I traveled in the past seem to be connected at a certain point in time. Take this study as an example, the gut microbes are super-complex living organisms, and the intestine is like a miniature ecosystem. To determine how they communicate with the brain and verify the types of bacteria that are actually involved, a different point of view can be initiated if the underlying theoretical mechanism is considered from an ecological perspective.”

《Nature》自然雜誌是科學領域的頂尖期刊，在2021年6月最新發佈的期刊引用指標（JCR: Journal Citation Report）的綜合科學領域（Multidisciplinary Sciences） 排名第一，最新影響因子（Impact factor）為49.962。《Nature》的投稿要求非常高，吳偉立表示，而大腦、腸道的連結與溝通機制，以及腸道中微生物如何影響大腦神經反應，研究議題也相當複雜，實驗難度高，這為時6年的研究加上投稿上稿的過程，從美國到臺灣：包含3年在美國加州理工學院以及3年在成功大學的研究，能做出這份完整的研究十分不容易！

Nature is a top scientific journal. In June 2021, it was ranked first place in the category of multidisciplinary sciences in the latest Journal Citation Report, and its current impact factor is 49.962. Nature has extremely stringent review process. Wu stated that the research topic, which involves communication mechanisms between the brain and the intestine, as well as how the microbes in the intestine affect the brain’s neurological response, is fairly complicated, which in turn leads to difficult experiments. The process of research and manuscript submission took 6 years and carried on from United States to Taiwan, including 3 years of research at the California Institute of Technology and another 3 years at NCKU. Conducting a complete study like this is no easy feat.

在美國加州理工學院擔任博士後研究的6年半的過程中，先是遭遇知名神經生物學家 Paul Patterson指導教授腦癌驟逝，研究不得不中途更換主題與轉換研究領域，到了Sarkis Mazmanian微生物與免疫學實驗室，雖然實驗室在發展神經科學方向，但是仍然受限於儀器設備的限制，例如實驗室沒有老鼠腦部立體定位儀，因此吳偉立的實驗初期是在缺乏專業儀器輔助與研究人力稀少的狀態下持續努力前進，也練就一身盡己所能盡量問人、想盡辦法找資源的功夫，充分感受到出外廣泛結交朋友的好處。

During his 6 and a half years as a postdoctoral researcher at the California Institute of Technology, Wu’s research on maternal immune response and autism was hampered by the death of his postdoctoral mentor, Professor Paul Patterson, a pioneer and renowned neurobiologist, due to brain tumor. He switched to a different subject and research area in the middle of his postdoctoral research. He relocated to the Sarkis Mazmanian Lab of Microbiology and Immunology, and although the research focus of the laboratory has shifted to neuroscience, it still had some limitations. For example, the laboratory at that time did not have a stereotaxic rig for mice. In the early stage of his experiment, Wu pressed on without the assistance of equipment and with scarce manpower. This sharpened his communication skills to find the resources, allowing him to fully understand the benefits of having a broad social network in academia.

年輕學者發表重要研究成果，成大老中青三代學者共同開創臺灣新科學研究態度

Young scholars publishing important research results is the outcome of a new research attitude in Taiwan, established by three generations of scholars working together. Left to right: Tzu-Hsuan Yao, Yuan-Yuan Lin, Chia-Wei Liou, Tzu-Ting Lai.

來到成大後個人研究室剛起步不久，吳偉立感謝成大對於新進教師的友善、支持、栽培與包容，以及劉嘉瑋、賴姿廷、林元元、姚子宣這4位各具風格的成大研究生，分別在不同階段，付出額外的時間與心力，一同冒險犯難，逐步壯大研究陣容，也十分感謝美國加州理工學院生物及生物工程學院 Sarkis Mazmanian 教授跨海提供協助。

With Wu’s lab still in its growing following his third year at NCKU, Wu was grateful for the kindness, support, cultivation, and tolerance that NCKU showed toward junior faculties. He was also thankful to Chia-Wei Liou, Tzu-Ting Lai, Yuan-Yuan Lin, and Tzu-Hsuan Yao, four NCKU students, each with their own research style, who invested extra time and effort at different stages and braved through difficulties together, gradually expanding the research team. Wu also expressed his gratitude to the Professor Sarkis Mazmanian in the Division of Biology and Biological Engineering at California Institute of Technology for enormous contributions overseas.

吳偉立表示，目前透過動物實驗找到糞腸球菌，證實糞腸球菌有助減輕小鼠壓力並調控其社交行為，學界也有部分研究支持這樣的論點，但這隻細菌的抗藥性很強，若能成功分離出降低壓力賀爾蒙及增加社交行為的糞腸球菌成分，未來應用價值會比較高。團隊將持續投入系列研究，找出糞腸球菌究竟是透過何種方式或其代謝物質來影響宿主的壓力反應。

According to Wu, the current discovery of E. faecalis through animal experiments has verified that E. faecalis can help reduce stress in mice and regulate their social behavior. However, E. faecalis also has extremely strong antibiotic resistance. If the components or metabolites of E. faecalis that reduce stress hormones and increase social behavior can be successfully isolated, they will have high application value. The team will continue a series of studies to determine how E. faecalis or its metabolites affect the host’s stress responses.

成大破解腸道菌透過腦部神經迴路 抑制壓力賀爾蒙影響社交行為

NCKU solves the mystery of how a gut bacterium inhibits stress hormones and affects social behavior through brain neural circuits

文／成大新聞中心　圖／成大生理所吳偉立實驗室

成大生理所助理教授吳偉立研究團隊發現腸道細菌「糞腸球菌」透過腦部神經迴路，抑制壓力賀爾蒙影響社交行為。

A research team led by Assistant Professor Wu Wei-Li in the Department of Physiology, NCKU, discovered that a gut bacterium, Enterococcus faecalis, inhibits stress hormones and affects social behavior through brain neural circuits.

本研究首先證明完全無菌鼠（germ-free mice；全身都沒有任何共生細菌）以及成年時期接受抗生素處理細菌後的老鼠，在面對同種個體時的社交互動行為表現量均較低；然而，其血液中的壓力賀爾蒙-皮質酮濃度則較高。研究團隊進一步利用藥物阻斷皮質酮訊息路徑，或是經由手術摘除腎上腺以阻斷產生皮質酮的主要來源時，發現降低壓力賀爾蒙-皮質酮可以恢復無菌鼠的社交行為。

The research first verified that germ-free mice (no symbiotic bacteria in their bodies) and mice that received antibiotics for bacterial elimination in adulthood had lower levels of social behavior with individuals of the same species. However, the levels of the stress hormone in their blood, corticosterone, were higher. When the research team used drugs to block the signaling pathway of corticosterone, or surgically removed the adrenal glands to block the main source of corticosterone production, they found that the social behavior of germ-free and antibiotic-treated mice was restored.

研究亦發現在腦部特定區域精準剔除皮質酮接受器-葡萄糖皮質素受體，或是利用化學遺傳學抑制促腎上線皮質激素釋放激素神經元（分泌皮質酮的上游路徑），皆可降低抗生素處理後小鼠的壓力賀爾蒙，以及增加小鼠的社交行為。相反的，在具有正常腸道共生細菌的小鼠身上，以化學遺傳學方式促進促腎上線皮質激素釋放激素神經元，或是直接刺激特定腦區壓力反應神經元，壓力增加後皆會造成小鼠社交行為缺損。

Their study also discovered that the precise removal of corticosterone receptors—glucocorticoid receptor—in specific brain areas, or the use of chemogenetic to inhibit corticotropin-releasing hormone neurons (the upstream pathway of corticosterone secretion), can reduce stress hormone levels in mice after antibiotic treatment and increase their social behavior. By contrast, in mice with normal gut microbiota, using chemogenetic to activate corticotropin-releasing hormone neurons, or directly stimulating stress-responsive neurons in specific brain areas, will cause impairment in the social behavior following stress.

研究團隊發現在抗生素處理過後的老鼠腸道中給予一種特定腸道細菌「糞腸球菌」，將可以降低其壓力賀爾蒙以及增加其社交行為，成功破解腸道與大腦的摩斯密碼，證實了腸道細菌調控動物的壓力反應與社交行為。

The research team discovered that providing a specific gut bacterium, E. faecalis, in the intestines of mice treated with antibiotics can reduce their stress hormones and increase their social behavior, successfully cracking the secret code of the intestine and brain. This verified that gut bacteria regulate the stress response and social behavior of animals.

本研究參與協助完成研究的台灣學生包括：成功大學基礎醫學所學生劉嘉瑋（第三作者）、成功大學生理所學生賴姿廷（第五作者）、林元元（第十二作者）、姚子宣（第十三作者）等四位成功大學學生。本研究同時聯合加州理工學院 Rustem Ismagilov 教授及 Viviana Gradinaru 教授及其團隊跨國合作。

The Taiwanese students who participated in this study to assist in its completion included four NCKU students: Chia-Wei Liou (third author) of the Institute of Basic Medical Sciences, Tzu-Ting Lai (fifth author), Yuan-Yuan Lin (twelfth author), and Yao Tzu-Hsuan (thirteenth author) of the Department of Physiology. The study also involved a transnational collaboration with Professors Rustem Ismagilov and Viviana Gradinaru as well as their teams at the California Institute of Technology.