This material is based on the works supported by Nanyang Assistant Professorship (NAP, M4080740), Agency for Science, Technology and Research (A*STAR) Public Sector Research Funding (PSF, M4070190), A*STAR-JST (The Japan Science and Technology Agency) joint grant (M4070198), and Singapore Ministry of Education (MOE2013-T2-2-049). The authors would like to thank Mr. Roger Tan Kay Chia, Prof. Low Kin Huat, Mr. Poon Kee Chun, Mr. Chew Hock See, Mr. Lam Kim Kheong and Dr. Mao Shixin at School of MAE for their support in setting up and maintaining the research facilities. The authors thank Prof. Michel Maharbiz (U.C. Berkeley) his advice and discussion, Prof. Kris Pister and his group (U.C. Berkeley) for their support in providing the GINA used in this study.

1. 연구 동물 <ol><li> 후면 개별 Mecynorrhina의 torquata 딱정벌레 나무 펠렛 침구 별도의 플라스틱 용기에 (6cm, 8g). </li><li> 각 딱정벌레에게 설탕 젤리 (12 ml)을 3 일마다 한 잔을 공급. </li><li> 각각 25 ° C, 60 %로 사육 실내의 온도와 습도를 유지한다. </li><li> 얇은 와이어 전극을 주입하기 전에 각 딱정벌레의 비행 능력을 테스트합니다. <ol><li> 부드럽게 공기에 딱정벌레를 throw합니다. 딱정벌레는 5 연속 시험에 대한 이상 10 초 동안 비행 할 수있는 경우, 딱정벌레 일반 비행 기능을 가지고 결론 및 후속 비행 실험을 위해 그것을 사용한다. 어두워하기 위해 방에있는 모든 조명을 끄고, 딱정벌레를 탈환합니다. 이 비행을 종료 할 딱정벌레가 발생합니다. 참고 : 공기 중으로 방출 할 때 딱정벌레가 자발적으로 멀리 비행을 시작합니다. 예컨대도 1 (16 × 8 × 4m에 도시 한 바와 같이 큰 폐 공간에서 비행 실험을 실시하는 것이 좋다 <su의 모션 캡처 공간 P&gt; 3 12.5 X 8 × 4m 3), 공중에서 회전 할 때 비행 딱정벌레 이동 매우 빠르게 (약 3-5m / 초)과 큰 호를 그립니다. </li></ol></li></ol> 2. 전극 주입 <ol><li> 1 분 13,16,20-24에 대한 CO 2로 채워진 플라스틱 용기에 배치하여 벌레를 마취. </li><li> 10 초 동안 온수에 침지하여 치과 용 왁스를 발라. 나무 블록에 마취 딱정벌레를 놓고 연화 치과 왁스로 고정시킨다. 치과 왁스는 자연적으로 냉각하고 몇 분 이내에 굳은. </li><li> 25mm의 길이로 잘라 절연은 전선 (127 μm의 맨 직경, 퍼플 루오로 코팅 된 178 μm의 직경) 주입을위한 얇은 와이어 전극을 사용할 수 있습니다. </li><li> 각 와이어의 양 단부에 상기 절연체를 화염에 의해 베어 실버 3mm 노출. </li><li> SMA를 만들 뾰족한 가위를 이용하여 딱정벌레의 큐티클의 표면을 해부하다metepisternum (그림 2C)에 약 4 × 4mm의 LL 창. 참고 :도 2c에 도시 된 바와 같이 부드러운 갈색 색상의 표피가 다음 노출 - 전자. 3AX 근육은 부드러운 표피 아래에 있습니다. </li><li> 피어스 개의 구멍 (도 2D) 사이에 2mm의 거리 곤충 핀 (크기 00)를 사용하여 노출 된 갈색 표피 두 구멍. </li><li> 주의 깊게 관통 구멍 (단계 2.4에서 제조 한 액티브 한 귀환 전극을 포함)이 와이어 전극을 넣고, 3 mm의 깊이로 각 3AX 근육로 이식. </li><li> 이식 된 전극을 고정하고 구멍에 녹은 밀랍을 놓아 연락처 및 단락을 방지하기 위해 장소에 개최합니다. 필요한 경우, 뜨거운 납땜 인두의 끝과 밀랍을 터치하여 표피를 통해 밀랍을 리플 로우. 밀랍 빠르게 굳은 및 주입을 강화. 참고 : 주입이 올 경우 무의 딱지 날개를 확인하려면제작 전기 자극 중 3AX 근육의 움직임을 관찰하기 위해 해제 될 수있다. </li></ol> 3. 무선 배낭 조립 주 : 배낭 내장 무선 마이크로 4 층 FR-4 기판 상에 (1.6 X 1.6 cm 2)로 구성되었다. 배낭은 리튬 폴리머 microbattery (3.7 V, 350 ㎎, 10 MAH)에 의해 구동되었다. 전지를 포함하는 배낭의 총 질량은 비틀 (10 g 체중의 30 %)의 페이로드 용량 미만 1.2 ± 0.26 g이었다. 배낭은 무선 통신을 수신하기 위해 미리 프로그램과 2 개의 출력 채널이었다. <ol><li> 앞가슴 등판의 표면을 청소 양면 테이프를 사용하여 (표피의 왁스 층을 제거). 그런 다음, 양면 테이프의 조각을 가진 딱정벌레의 앞가슴 등판의 배낭을 연결합니다. </li><li> 배낭의 출력에 이식 된 전극의 끝을 연결합니다. </li><li> fo를 마커를 생성하기 위해 microbattery 주위에 역 반사 테이프를 감싸R 모션 캡쳐 카메라를 검출한다. </li><li> 되 반사 테이프가 모션 캡쳐 카메라에 의해 검출 할 수 있으므로, 양면 테이프의 조각을 사용하여 가방의 상부에 microbattery 첨부. </li></ol> 4. 무선 제어 시스템 주의 : 이러한 경우에, 용어 무선 제어 장치는 원격 제어기를위한 수신기를 포함하는, 커스텀 비행 제어 소프트웨어를 실행하기위한 노트북 컴퓨터, 기지국, 배낭 및 모션 캡쳐 시스템. <ol><li> USB 포트를 통해 랩탑 컴퓨터의 리모트 컨트롤러의 기지국 수신기에 연결한다. </li><li> 모션 캡쳐 시스템에서 스위치 및 이더넷 포트를 통해 노트북 컴퓨터에 연결합니다. </li><li> 완전 모션 캡쳐 공간을 커버 (모션 캡쳐 시스템의 공급 회사가 제공하는) 보정 지팡이를 흔들며 부피 보정을 수행한다. <ol><li> 노트북의 바탕 화면에서 모션 캡쳐 소프트웨어를 엽니 다. 클릭 한 박사AG는 &quot;자원&quot;패널의 &quot;시스템&quot;메뉴에서 모든 카메라를 선택합니다. </li><li> 은 &quot;3D 관점&quot;메뉴를 클릭하고 카메라보기로 변경 &quot;카메라&quot;를 선택합니다. 교정 설정을 표시하는 &quot;도구&quot;패널에서 &quot;카메라&quot;탭을 클릭합니다. 카메라에서 잡음을 제거하기 위해 &quot;카메라 마스크 만들기&quot;메뉴에서 &quot;시작&quot;버튼을 클릭 잡음이 파란색 마스크 후 다음 &quot;정지&quot;. </li><li> 클릭하고 &quot;지팡이&quot;메뉴와 &quot;카메라&quot;탭에서 &quot;L-프레임&quot;메뉴에서 &quot;5 마커 지팡이 &amp; L-프레임&quot;을 선택합니다. 2500에 &quot;지팡이 카운트를&quot;설정은 &quot;교정 카메라&quot;메뉴의 &quot;시작&quot;을 클릭하고 전체 모션 캡처 공간을 통해 교정 지팡이를 흔들어. 막대 수가 2,500에 도달하면 교정 프로세스가 중지됩니다. </li><li> ( &quot;도구&quot;패널의 &quot;카메라&quot;탭의 하단에있는) 이미지 오류가보다 높은 0.3 F 인 경우 교정 과정을 반복또는 카메라. 교정 후, 모션 캡처 공간의 중앙에 바닥에 지팡이를 넣어 모션 캡쳐 공간의 원점을 설정하려면 &quot;설정 볼륨 기원&quot;메뉴의 &quot;시작&quot;을 클릭합니다. </li></ol></li><li> 모션 캡쳐 공간에서 사용자에 의해 흔들어 마커의 이동 경로를 기록하고 마커가 검출 및 추적 여부를 확인하기 위해 테스트 더미를 이용하여 모션 캡쳐 시스템의 범위를 확인한다. 마커가 자주 검색하는 동안 손실 된 경우 더미 테스트가 성공할 때까지, 볼륨 교정을 반복합니다. <ol><li> &quot;도구&quot;패널에서 &quot;캡처&quot;탭을 클릭 한 다음 그 궤적을 기록 전체 모션 캡처 공간을 통해 샘플 마커를 흔들며하기 전에 &quot;캡처&quot;메뉴의 &quot;시작&quot;. </li><li> 기록 후, 클릭 마커의 위치를​​ 재구성하고 기록의 품질을 확인하기 &quot;를 재구성 파이프 라인을 실행합니다.&quot; </li></ol></li><li> microb의 단자에 연결attery 배낭의 전원 핀 (단계 3.4에서 배낭에 부착). </li><li> 랩톱과 맞춤 비행 제어 소프트웨어를 사용하여 배낭 사이의 무선 통신을 테스트한다. 소프트웨어의 &quot;시작&quot;명령을 클릭하고 표시된 연결 상태를 확인합니다. </li></ol> 5. 무료 비행 실험 <ol><li> 16 × 8 × 4m (3)을 측정하는 비행 분야에서 자유 비행 실험을 실시하고 있습니다. </li><li> 입력 비행 제어 소프트웨어 (전압, 펄스 폭, 주파수 및 자극 지속 시간)에 해당하는 파라미터. 참고 : 데모, 우리는 3 밀리로 3 V까지 펄스 폭의 전압을 고정하고, 자극 지속 시간이 1 초에 60 내지 100 Hz로 주파수를 변경. <ol><li> 은 &quot;전압&quot;상자에 3 V에 대한 소프트웨어 화면, 유형 3은 &quot;자극 기간&quot;상자에 1,000 밀리의 &quot;펄스 폭&quot;상자에 3 밀리 3 1,000, 및에서 Hz 단위 원하는 주파수에서 &quot; 주파수 &quot;박스 오n은 명령 창을여십시오. </li></ol></li><li> 이 비행 경기장 내에서 자유롭게 비행 할 수 있도록 공중으로 배낭에 장착 된 딱정벌레를 놓습니다. 딱정벌레는 모션 캡쳐 공간에 진입 할 때 수동 자극을 발생시킨다. 를 눌러 원격에서 해당 명령 단추 (왼쪽 또는 오른쪽)는 딱정벌레의 왼쪽이나 오른쪽에있는 대상 근육을 자극합니다. 주의 :이 버튼이 눌러지면, 랩톱에서 실행되는 비행 제어 소프트웨어 명령을 생성하고, 배낭으로 보낸다. 배낭은 (왼쪽 또는 오른쪽) 관심의 근육에 전기 자극을 출력한다. </li><li> 자극하는 동안 실시간으로 딱정벌레의 반응을 관찰하고 3D 그래프 소프트웨어를 사용하여 데이터를 재구성. <ol><li> 은 &quot;비틀 디스플레이&quot;윈도우의 데이터 목록에 기록 된 시험 중 하나를 선택하고 분석 폴더에 그 재판의 데이터를 복사하고 3 차원 그래프 모듈을 실행하는 &quot;수출 팬더&quot;를 클릭합니다. </li><li> 눌러 &quot;N&quot;키보드 기록 궤도에 자극 신호를 결합한다. I를 눌러 강조 표시된 자극 기간이 딱정벌레의 궤도를 표시합니다. </li></ol></li></ol>

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C., Herrera, M., Bustamante, M., Shingiro, A., Bowen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+Stimulus+Parameters+for+Directed+Locomotion+in+Madagascar+Hissing+Cockroach+Biobot.">Effective Stimulus Parameters for Directed Locomotion in Madagascar Hissing Cockroach Biobot.</a> PLoS ONE. 10 (8), e0134348 (2015).</li><li>Zhaolin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preliminary+study+of+motion+control+patterns+for+biorobotic+spiders.">A preliminary study of motion control patterns for biorobotic spiders.</a> , 128-132 (2014).</li><li>Feng, C., Chao, Z., Hao Yu, C., Sato, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect-machine+hybrid+robot:+Insect+walking+control+by+sequential+electrical+stimulation+of+leg+muscles.">Insect-machine hybrid robot: Insect walking control by sequential electrical stimulation of leg muscles.</a> , 4576-4582 (2015).</li><li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Biological+Micro+Actuator:+Graded+and+Closed-Loop+Control+of+Insect+Leg+Motion+by+Electrical+Stimulation+of+Muscles.">A Biological Micro Actuator: Graded and Closed-Loop Control of Insect Leg Motion by Electrical Stimulation of Muscles.</a> PLoS ONE. 9 (8), e105389 (2014).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromechanism+Study+of+Insect-Machine+Interface:+Flight+Control+by+Neural+Electrical+Stimulation.">Neuromechanism Study of Insect-Machine Interface: Flight Control by Neural Electrical Stimulation.</a> PLoS ONE. 9 (11), e113012 (2014).</li><li>Tsang, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+Split-Ring+Electrode+for+Insect+Flight+Biasing+Using+Multisite+Neural+Stimulation.">Flexible Split-Ring Electrode for Insect Flight Biasing Using Multisite Neural Stimulation.</a> IEEE Trans. Biomed. Eng. 57 (7), 1757-1764 (2010).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> J. Insect Physiol. 46 (4), 439-442 (2000).</li><li>Cooper, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia,+Analgesia,+and+Euthanasia+of+Invertebrates.">Anesthesia, Analgesia, and Euthanasia of Invertebrates.</a> ILAR Journal. 52 (2), 196-204 (2011).</li><li>Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect neurophysiological techniques. , (2012).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA guidelines for the euthanasia of animals. , (2013).</li><li>Heath, B., West, G., Heard, D., Caulkett, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobile+Inhalant+Anesthesia+Techniques.">Mobile Inhalant Anesthesia Techniques.</a> in Zoo Animal and Wildlife Immobilization and Anesthesia. , 75-80 (2008).</li><li>Mischiati, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+models+direct+dragonfly+interception+steering.">Internal models direct dragonfly interception steering.</a> Nature. 517 (7534), 333-338 (2015).</li><li>Kutsch, W., Berger, S., Kautz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Manoeuvres+in+Free-Flying+Locusts:+Two-Channel+Radio-Telemetric+Transmission+of+Muscle+Activity.">Turning Manoeuvres in Free-Flying Locusts: Two-Channel Radio-Telemetric Transmission of Muscle Activity.</a> J. Exp. Zoolog. Part A Comp. Exp. Biol. 299 (2), 139-150 (2003).</li><li>Wang, H., Ando, N., Kanzaki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+Control+of+Free+Flight+Manoeuvres+in+a+Hawkmoth,+Agrius+Convolvuli.">Active Control of Free Flight Manoeuvres in a Hawkmoth, Agrius Convolvuli.</a> J. Exp. Biol. 211 (3), 423-432 (2008).</li><li>Sato, H., Maharbiz, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+the+remote+radio+control+of+insect+flight.">Recent developments in the remote radio control of insect flight.</a> Front. Neurosci. 4, (2010).</li><li>Tien Van, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flight+behavior+of+the+rhinoceros+beetle+Trypoxylus+dichotomus+during+electrical+nerve+stimulation.">Flight behavior of the rhinoceros beetle Trypoxylus dichotomus during electrical nerve stimulation.</a> Bioinsp. Biomim. 7 (3), 036021 (2012).</li><li>Sane, S. P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+flight+force+by+a+flapping+wing:+lift+and+drag+production.">The control of flight force by a flapping wing: lift and drag production.</a> J. Exp. Biol. 204 (15), 2607-2626 (2001).</li><li>de Croon, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+aerodynamics+and+autonomy+of+the+DelFly.">Design, aerodynamics and autonomy of the DelFly.</a> Bioinsp. Biomim. 7 (2), 025003 (2012).</li><li>Ma, K. Y., Chirarattananon, P., Fuller, S. B., Wood, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Flight+of+a+Biologically+Inspired,+Insect-Scale+Robot.">Controlled Flight of a Biologically Inspired, Insect-Scale Robot.</a> Science. 340 (6132), 603-607 (2013).</li></ol>

The authors declare that there are no conflicts of interest.

이 실험의 신뢰도에 영향을 미치는, 상기 주입 공정은 중요하다. 전극은 3mm 비틀 (주변 근육과의 접촉을 회피)의 크기에 따라 이하의 깊이에서 근육에 삽입되어야한다. 전극이 근처의 근육을 터치하면 바람직하지 않은 모터 행동과 행동은 주변 근육의 수축으로 인해 발생할 수 있습니다. 두 개의 전극이 아니라 더 단락이 발생하지 않도록 정렬되어야합니다. 용융 및 납땜을 사용하여 밀랍을 리플 ...

An insect-machine hybrid system, often referred to as a cyborg insect or biobot, is the fusion of a living insect platform with a miniature mounted electronic device. The electronic device, which is wirelessly commanded by a remote user, outputs an electrical signal to electrically stimulate neuromuscular sites in the insect via implanted wire electrodes to induce user desired motor actions and behaviors. In the early stages of this research field, researchers were limited to conducting wireless recording of the muscular action of an insect, using simple analog circuits comprised of surface-mounted components1-3. The development of system-on-a-chip technology with radio frequency functionality enabled not only the wireless recording of neuromuscular signals but also the electrical stimulation of the neuromuscular sites in living insects. At present, a built-in radio microcontroller is small enough to be mounted on living insects without causing any obstructions to their locomotion4-13.
The development of the built-in radio microcontroller allows researchers to determine electrical stimulation protocols to induce desired motor actions to control the locomotion of the insect of interest. On the ground, researchers have demonstrated walking control by stimulating the neuromuscular sites of cockroaches4,12,14, spiders15, and beetles16,17. In the air, the initiation and cessation of flight were achieved using different methods such as the stimulation of the optic lobes (the massive neural cluster of a compound eye) in beetles7,9 and brain sub-regions in bees18, whereas turning control has been demonstrated by stimulating the antennae muscles and nervous system of the abdomens in moths11,19 and the flight muscles of beetles7,9,13. In most cases, a built-in radio microcontroller was integrated on a custom-designed printed circuit board to produce a miniature wireless stimulator (backpack), which was mounted on the insect of interest. This allows wireless electrical stimulation to be applied to a freely walking or flying insect. Such a microcontroller-mounted insect is what is referred to as an insect-machine hybrid system. 
This study describes the experimental protocols for building an insect-machine hybrid system, wherein a living beetle is employed as the insect platform, and instructs on how to operate the robot and test its flight control systems. The third axillary sclerite (3Ax) muscle was chosen as the muscle of interest for electrical stimulation and demonstration of left or right turning control13. A pair of thin silver wire electrodes was implanted in both the left and right 3Ax muscles. Moreover, a backpack was mounted on the living beetle. The other ends of the wire electrode were connected to the output pins of the microcontroller. The backpack was small enough for the beetle to carry in flight. Thus, this allows an experimentalist to remotely stimulate the muscle of interest of an insect in free flight and investigate its reactions to the stimulations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mecynorrhina torquata beetle</td>
			<td>Kingdom of Beetle Taiwan</td>
			<td></td>
			<td>10 g, 8 cm, pay load capacity is 30% of the body mass 
			Aproval of importing and using by Agri-Food and Veterinary Authority of Singapore (AVA; HS code: 01069000, product code: ALV002).</td>
		</tr>
		<tr>
			<td>Wireless backpack stimulator</td>
			<td>Custom</td>
			<td></td>
			<td>TI CC2431 micocontroler 
			The board is custom made based on the GINA board from Prof. Kris Pister&#8217;s lab. The layout of GINA board can be found at&#160;&#160;&#160; https://openwsn.atlassian.net/wiki/display/OW/GINA</td>
		</tr>
		<tr>
			<td>Wii Remote control</td>
			<td>Nintendo</td>
			<td></td>
			<td>Bluetooth remote control to send the command to the operator laptop</td>
		</tr>
		<tr>
			<td>BeetleCommander v1.8</td>
			<td>Custom. Maharbiz group at UC Berkeley and Sato group at NTU</td>
			<td></td>
			<td>Establish the wireless communication of the backpack and the operator laptop. Configure the stimulus parameters and log the positional data. Visualize the flight data.</td>
		</tr>
		<tr>
			<td>GINA base station</td>
			<td>Kris Pister group at UC Berkeley</td>
			<td></td>
			<td>TI MSP430F2618 and AT86RF231</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T160</td>
			<td>8 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Motion capture system</td>
			<td>VICON</td>
			<td>T40s</td>
			<td>12 cameras for a flight arena of 12.5 x 8 x 4 m</td>
		</tr>
		<tr>
			<td>Micro battery</td>
			<td>Fullriver</td>
			<td>&#160;201013HS10C&#160;</td>
			<td>3.7V, 10 mAh</td>
		</tr>
		<tr>
			<td>Retro reflective tape</td>
			<td>Reflexite</td>
			<td>V92-1549-010150</td>
			<td>V92 reflective tape, silver color</td>
		</tr>
		<tr>
			<td>PFA-Insulated Silver Wire&#160;</td>
			<td>A-M systems</td>
			<td>786000</td>
			<td>127 &#181;m bare, 177.8 &#181;m coated, 3 mm bare silver flame exposed at tips</td>
		</tr>
		<tr>
			<td>SMT Micro Header&#160;</td>
			<td>SAMTEC</td>
			<td>FTSH-110-01-L-DV</td>
			<td>0.3 x 6 mm, bend to make a 3 mm long slider to secure the electrode into the PCB header.</td>
		</tr>
		<tr>
			<td>Beeswax</td>
			<td></td>
			<td></td>
			<td>Secure the electrodes</td>
		</tr>
		<tr>
			<td>Dental Wax</td>
			<td>Vertex</td>
			<td></td>
			<td>Immobilize the beetle</td>
		</tr>
		<tr>
			<td>Insect pin</td>
			<td>ROBOZ</td>
			<td>RS-6082-30</td>
			<td>Size&#160; 00; 0.3mm Rod diameter; 0.03 mm tip width; 38 mm Length&#160; 
			Make electrode guiding holes on cuticle</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>DUMONT</td>
			<td>RS-5015</td>
			<td>Pattern #5; .05 X .01mm Tip Size; 110mm Length 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>ROBOZ</td>
			<td>RS-5620</td>
			<td>Vannas Micro Dissecting Spring Scissors; Straight; 3mm Cutting Edge; 0.1mm Tip Width; 3&#34; Overall Length&#160; 
			Dissecting and implantation</td>
		</tr>
		<tr>
			<td>Potable soldering iron</td>
			<td>DAIYO</td>
			<td>DS241</td>
			<td>Reflow beeswax</td>
		</tr>
		<tr>
			<td>Hotplate&#160;</td>
			<td>CORNING</td>
			<td>PC-400D</td>
			<td>Melting beeswax and dental wax</td>
		</tr>
		<tr>
			<td>Flourescent lamp</td>
			<td>Philips</td>
			<td>TL5 14W</td>
			<td>Light the entire flight arena with 30 panels (60 x 60 cm2). Each panel has 3 lamps. 
			14 W, 549 mm x 17 mm&#160;</td>
		</tr>
	</tbody>
</table>

insect-machine hybrid system: remote radio control of a freely flying beetle (mercynorrhina torquata)

무선 호환 디지털 전자 기기의 상승은 비행 곤충의 행동을 연구하기위한 작은 무선 신경 근육 레코더 및 자극의 사용을 묻는 메시지가있다. 이 기술은이 프로토콜에서 설명 살아있는 곤충의 플랫폼을 사용하여 곤충 기계 하이브리드 시스템의 개발을 가능하게한다. 더욱이,이 프로토콜은 시스템 구성 및 풀려 곤충의 비행 근육의 기능을 평가하기위한 자유 비행 실험 절차를 나타낸다. 데모를 위해, 우리는 제어 및 비행 딱정벌레의 왼쪽 또는 오른쪽으로 회전을 달성하기 위해 세 번째 겨드랑이 sclerite (3AX) 근육을 대상으로. 얇은 실버 와이어 전극은 딱정벌레의 각 측면에 3AX 근육에 이식했다. 이들은 무선 배낭의 출력에 연결되어 (즉, 신경 근육 전기 자극)는 딱정벌레의 앞가슴 등판에 장착. 근육은 (왼쪽 또는 오른쪽) 자극 측 교대 또는 stimulatio를 변화시킴으로써 자유 비행 자극했다n 개의 주파수. 근육이 자극 증가 주파수에 대한 등급 화 반응을 나타냈다 때 딱정벌레는 동측 측에 돌렸다. 주입 공정 및 3 차원 모션 캡쳐 카메라 시스템의 볼륨 조정을 각각 근육 손상 및 마커 트랙의 손실을 방지하기 위해주의 깊게 수행되어야한다. 무료로 비행에 대한 관심의 비행 근육의 기능을 공개하는 데 도움이 방법은, 곤충 비행을 연구하는 것이 매우 유리하다.

전극 주입 절차는 그림 2에 제시되어 얇은 실버 와이어 전극 근육 (도 2D - 전자)의 부드러운 표피에 피어싱 작은 구멍을 통해 딱정벌레의 3AX 근육에 이식했다.. 이 부드러운 표피는 metepisternum의 앞쪽 부분을 제거한 후 바로 basalar 근육의 apodema 위에서 발견된다 (도 2 차원을 - 다). 전극은 다음 밀랍 (도 2F)?...

Watch this Scientific Journal Video about Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata at JoVE.com

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

곤충 - 기계 하이브리드 시스템 : 자유롭게 비행 비틀의 원격 무선 제어 (<em&gt; Mercynorrhina의 torquata</em&gt;)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

insect-machine-hybrid-system-remote-radio-control-freely-flying

Research

JoVE Journal

Neuroscience

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

12.1K 조회수.  Nanyang Technological University. This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

비디오: Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

3D 신경 줄기 세포 문화 번역하는 단백질 : 하이브리드 시각화 방법론

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

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신경과학

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

자유롭게 수영 약하게 전기 생선의 장기 행동 추적

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

매우 낮은 밀도에 대한 단순화 된 방법, 장기 차 해마 신경 세포 문화

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

뉴런의 유전자 인코딩 된 부 자연스러운 아미노산을 사용하여 신경 세포 단백질의 광 제어

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

절제된 자유 비행 꿀 꿀벌의 Neuropharmacological 조작,<em&gt; API를 mellifera이</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

새로운 방법으로 자유롭게 움직이는 신생아 쥐의 뇌파 기록

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

자유롭게 이동 Piglets에서 비 침 투 적인 뇌 파 기록

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

자유롭게 행동 하는 쥐에 장기 감각 충돌

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

자유롭게 움직이는 마우스에서 이중 사이트 고밀도 레코딩을 위한 회수 가능한 광실리콘 프로브 및 Tetrode가 있는 하이브리드 마이크로드라이브 시스템

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

자유롭게 수영 물고기에서 이동 식 Tetrodes에 의해 뉴런의 무선 전기 생리적 기록

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...