소개 생물이 위협 자극 환경의 변화에​​ 대응하는 행동의 다양한 전시해야합니다. 자율 신경계의 반응은 잘 공감 신경계는 생리학 내부 환경 (.; 니콜스 외, 2001 카펜터, 1976) readies있는 전투 또는 비행 응답으로 척추 동물에서 공부합니다. 그것은 유기체가 스트레스 반응을 조절하고 임박한 공격이나 환경 변화에 반응하는 동작을 변경할 수있는 생리적 변화하는 것입니다. 증거는 심장 혈관 시스템 - 단단히함으로써 내부 환경이 응답을 응답하고 구현할 수 있도록 호흡기 시스템 연결되었는지 보여줍니다. 그것은 잘 호흡기 및 심장 혈관 시스템의 자율 제어 산소 가용성 및 임박한 행동 반응에 필요한 특정 대상 조직에 영양분을 조절 수있는 것으로 알려져 있습니다. 척추 동물에서, 자율과 생리적 반응은 잘 발전이며 그것은 복잡한 무척추 동물은 비슷하게 개발 응답 시스템을 가지고 것이라는 가능성이 보이는군요 (. Schapker 외, 2002; Zavarzin 1941). 척추 동물과 무척추 동물이 체계적으로 매우 다르다하더라도, 고도로 발달한 무척추 동물 싸움이 또는 비행 방식으로 대응할 수있는 신속한 심혈관 및 호흡기 응답을 필요로합니다. 많은 무척추 동물의 경우, 교감 같은 생리적 반응 계량하실 수 있습니다. 1927까지 거슬러 올라가는, 아주 초기 연구의 대부분은 특별히 (; 올로브 1927; Zavarzin 1941 Alexandrowicz 1932) 일반적으로 arthropods를 사용하여, 무척추 동물에서 실시되었습니다. 크레이 피쉬는 빠른 행동의 광범위한뿐만 아니라 평가 및 환경 자극에 반응하는 능력을 전시하는 것으로 알려져 있습니다. 아주 초기 연구는 방위 허세의 즉각적이고 신속한 대응 (; 헉슬리, 1880; Shuranova 외, 2006;. Wiersma, 1961 Bethe, 1897)의 동정과 같은 반응을 언급했습니다. 비트와 리듬이 중추 신경계 (; 야마 기시와 히로세, 1997;. 야마 기 외, 1997; 윌켄스 1999 Alexandrowicz 1932)에 의해 제어되기 때문에 성인 왕새우의 마음이 neurogenic입니다. 일상적으로, 동물의 심장 박동 (HR)의 측정은 흥분과 내부 환경의 준비의 직접적인 측정을 제공합니다. 인식 위협 자극 (Listerman 외., 2000)이 드러나면 왕새우에서는 HR의 증가 방어 허세 동안 기록됩니다. 신경계 반응의 이러한 유형은 또한 가시 바다 가재 (야자와 야마, 2001)에 표시되었다. ventilatory 시스템은 또한 neuronally이 전문 부속 칭했다 scaphognathites의 펌핑 작업 (Mendelson, 1971)에 의해 아가미를 통해 산소 이해에 대한 책임 ventilatory 중앙 패턴 발생기 (VPG)으로 제어됩니다. 하나 scaphognathite은 앞쪽에 끝에 각 아가미의 챔버에있는과 리듬 운동 (Pasztor 1968 년)의 아가미를 통해 물을 그립니다. VPG은 심장과 마찬가지로, 여러 가지 요인에 의해 변조된 수 있습니다. 게에서 이전 작업, 암 매지 스터는 심장과 ventilatory 리듬이 명령 뉴런 (윌켄스 외., 1974)를 통해 중앙 제어에 의해 변경될 수 있습니다 보여줍니다. Cuadras, 1979, 1980,, 리 외, 2000;. Listerman 외뿐만 아니라, VPG의 활동은 내부 반응과 사회적 상호 작용이나 갑각류의 환경 변화 (Burmistrov 및 Shuranova, 1996 변화와 함께 변화하는 것으로 알려져 있습니다. , 2000; 맥마혼 및 윌켄스, 1983; Schapker 외, 2002;. Shuranova 외, 2002;. 윌켄스, 1976). 으로 크레이 피쉬에서 볼, ventilatory 활동 (VR)는 예상치 못한 외부 자극 (.; Shuranova 외, 2002. Shuranova 외, 1993)에 기록된 수있는 내부 상태와 VR의 변화에 따라 달라질 수 있습니다. HR과 VR 대책의 신뢰성과 타당성으로 인해, 요구하는 미래의 질문은 무한합니다. 이전 바와 같이, 작업은 사회적 상호 작용과 환경 교란하는 동안 자율 신경계의 반응을 조사하기 위해 수행되었습니다. 흥미롭게도, 많은 분야는 이러한 천연 호르몬 변화 및 ​​/ 또는 유기체에서 발생하는 다른 생리적 과정 동안 자율 반응 검사로 탐험으로 남아 있습니다. 향후 방향은 교미 및 후각 화학 리셉션 동안 교감과 같은 응답을 검사 수 있습니다. 가장 중요한 생리적 반응들은 내부적으로 자신을 준비하는 경우 동물들이 항상 behaviorally 응답하지 않기 때문에 행동 관찰에 비해 환경에 유기체의 반응에 큰 통찰력을 제공할 수 있습니다. 따라서 생리 측정은 유기체의 전체 변경 사항을 설명 수도 있습니다 유기체의 내부 상태를 평가하기위한 생물 학적 색인을 제공합니다. 방법 크레이 피쉬를 선을 시작하기 전에, 전선의 준비가 필요합니다. 충분히 왕새우에 넣고 충분히 (UFI, 모델 2991) 임피던스 검출기에 도달합니다. 2 개 절연 스테인레스 스틸 와이어 (AM 시스템, Carlsburg, WA 직경 0.005 인치 코팅 0.008 인치) 컷 등딱지에 삽입 될 두 전선의 끝을에서 화재로 절연 제를 (~ 0.5 mm)를 제거합니다. 동물의 외부에 노출된 와이어로 너무 절연을 제거하지 않도록주의가 짧은 circuiting 임피던스 녹음을 방지하기 위해 recoated해야합니다. 그런 다음, 집게를 사용하면 등딱지에 게재 위치에 대한 90 각도에서 전선의 구운 끝을 구부. 이 내부 장기에 손상을 할 수 있기 때문에 전선의 삽입 부분이 너무 긴 아니라는 것을 확인하십시오. 플라스틱 튜브 (전선을 보호하는)에 전선을 삽입 가능성에 대한, 그것은 느슨하게 함께 전선을 비꼬아하는 것이 좋습니다. 레코딩 마음과 환기 요금, 이상 반복 두 번째 단계와 전선의 두 세트는 플라스틱 튜브에 배치하기 전에 함께 느슨하게 꼰 수있다면. 와이어가 녹화되는 임피던스 감지기에 연결하는 혼동하지 않도록만큼 마음과 환기 율에 대한 전선을 지정해야합니다. 전선을 라벨이나 시작에서 다른 이상 한 쌍의 짧은자를하는 것이 좋습니다. feasibly 안전 왕새우를 와이어하려면, 그것은 사람 및 / 또는 왕새우 (그림 1)에 부상을 제거하는 서부 유럽 표준시 헝겊이나 종이 타올의 발톱과 다리를 (다시 떠나는하고 한쪽은 노출) 래핑하는 것이 좋습니다. <img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="그림 1" /> 그림 1. 왕새우 chelipeds과 다리의 포장. 젖은 종이 타월에 왕새우를 포장하는 것은 사람과 왕새우에 부상을 방지할 수 있습니다. 포장은 chelipeds와 다리를 움직일 수있는 왕새우의 능력을 방해합니다. 등딱지 두께가 각 왕새우와 과도한 힘으로 다릅니다부터 배선 과정을 시작하려면, 그것은 길 챔버에서 시작하는 것이 좋습니다하면 길 챔버에서 시작하여 따라서, 내부 장기에 손상을 일으킬 수있는 과도한 압력으로 왕새우를 죽일 가능성이 적습니다 흉부 챔버에 비해. 첫 번째 구멍은 아가미 챔버 (.; Schapker 외, 2002 년 즉, ventilatory 속도를 모니터링하는 prebranchial 챔버)의 주동이의 영역에있는 표피 아래에 삽입하는 와이어 충분한 메스 단 대형 벌금 포인트를 사용하여 이루어집니다. 큰 구멍이 과도한 hemolymph 손실을하며 제대로 밀봉되지 않으며 이것은 지정된 구멍에 남아 아니라 와이어의 확률을 증가합니다. 와이어가 자리에되면, 와이어에 접착제 (cyanoacrylate 에스테르) 및 가속기 (HobbyTown 미국, 렉싱턴, KY)의 작은 금액을 놓으십시오. 빠른 건조 접착제의 사용은 동물의 처리 부담을 줄일 수 있습니다. 이것은 왕새우에 독성이며 사망 원인이 있기 때문에 표피에 가속기만을 소량 곳으로주의하십시오. 즉시 종이 타월로 초과 가속기를 닦아하는 것이 좋습니다. 길 챔버에서 두 번째 와이어에 대해 이러한 단계를 반복합니다. 이 두 전선은 각 scaphognathite 운동 (그림 2) 중 강력한 정확한 임피던스 측정을 확보하기 위해 scaphognathite (호흡 기관)를 스팬하는 배치됩니다. 정확한 녹음을하려면 두 번째 와이어가 더 길 챔버 ​​외부와 흉부 챔버에 배치되지 않았는지 확인합니다. <img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="그림 2" /> 그림 2. 길 챔버의 스테인레스 스틸 와이어의 배치. 등딱지에 하얀 점들은 표피 아래에 배치하는 스테인레스 스틸 와이어에 대한 포인트를 나타냅니다. 전선은 동적 임피던스를 통해 scaphognathite 및 측정 운동을 스팬. 흉부 챔버의 배선 심장 박동에 대한 단계는 전선이 직접 주동이의 - 꼬리 배열에있는 심장과 스팬의 중심 (그림 3)을 통해 등의 등딱지 아래에 배치를 제외하고 위와 동일합니다. 이 배치는 각 심장 수축 (Listerman 외., 2001) 동안 강력한 정확한 임피던스 측정을 보장합니다. <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="그림 3" /> 그림 3. 흉부 챔버의 스테인레스 스틸 와이어의 배치. 등딱지에 하얀 점들은 표피 아래에 배치하는 스테인레스 스틸 와이어에 대한 포인트를 나타냅니다. 전선이 주동이의 - 꼬리 배열의 마음을 스팬. 이것은 동적 임피던스를 통해 심장 수축의 대책있게됩니다. 모두 4 개의 선이의 배치가 완료되면 (이스트먼, 5 분 건조 E에게 즉시 접착제를 혼합poxy)과 외투 각 지역뿐만 아니라 전선을 위해 구멍을. 전선 및 뚫고 구멍이 적절하게 적용되면, 왕새우의 뒤쪽에 플라스틱 튜브를 잡아 남은 에폭시를 사용합니다. 이것은 전선이 미래 handlings에있는 구멍에서 뽑아되지 않을 것임을 보장합니다. 에폭시 건조하는 데 몇 분 정도 걸리므로, 보유 또는 에폭시 더 이상 만져 끈적하지 않습니다 때까지 물없이 컨테이너에서 왕새우를 놓습니다. 에폭시가 건조되면, 과도한 화학 물질을 제거하고 수생 환경에 그들을 다시 곳으로 물속에 왕새우를 세척. 왕새우 건강을 보장하기 위해서는 이전에 제거되지 않은 화학 물질을 제거하는 약 한 시간 안에 물을을 변경하는 것이 좋습니다. 따라서 동물이 2로 3 일간 남아 있어야하는 실험하기 전에;로 이전 조사에서 명시된 바와 같이, 우리가 장기 처리는 3 일 (. 윌켄스 외, 1985) 24 H의 시간 동안 인해 스트레스 생리 측정을 바꿀지도 모르겠어 발견 . ECGs를 기록하려면, 왕새우에서 임피던스 감지기에 전선을 연결합니다. 심장 (측정 HR)를 걸쳐 두 전선이 두 전선이 scaphognathite (측정 VR)를 스팬에 대한 마찬가지로 하나의 임피던스 감지기에 붙어 있어야합니다. 임피던스 감지기가 두 스테인리스 와이어 사이의 역동적인 저항을 측정합니다. 이러한 신호는 PowerLab/4SP 인터페이스 (AD 악기)를 통해 PowerLab에 온라인 탐지기를 연결하여 기록됩니다. 모든 이벤트는 측정 및 PowerLab 차트 소프트웨어 버전 5.5.6 (AD 인 스트 루먼트, 호주)과 함께 조정됩니다. 시작하려면, 4 kHz에서에서 수집 속도를 설정합니다. 대형 deflections은 심장 박동과 환기 율을 나타내는 것입니다. 평활화 함수가 데이터 집합에 적용 할 수 각 HR / VR을 계산에 쉽게. 100 점이 smoothed있다면 (즉, 평균) 결과 추적은 심장이 뛰는을 나타내는 것입니다. 심장 요금 10 - S 간격이 지남에 따라 각 비트의 직접 건의에 의해 결정하고 분 (BPM) 당 비트로 변환할 수 있습니다.

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+heart+rate+to+measure+intrinsic+state+of+blind+cave+crayfish+during+social+interactions.">Use of heart rate to measure intrinsic state of blind cave crayfish during social interactions.</a> Comp. Bichem. Physiol. 127A, 55-70 (2000).</li><li>Listerman, L. R., Deskins, J., Bradacs, H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+within+male+crayfish:+social+interactions+and+effects+of+5-HT.">Heart rate within male crayfish: social interactions and effects of 5-HT.</a> Comp. Biochem. Physiol. 125, 251-263 (2000).</li><li>McMahon, B. R., Wilkens, J. L., Mantel, L., Bliss, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilation,+perfusion+and+oxygen+uptake.">Ventilation, perfusion and oxygen uptake.</a> Biology of Crustacea. 6, 289-372 (1983).</li><li>Mendelson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillator+neurons+in+crustacean+ganglia.">Oscillator neurons in crustacean ganglia.</a> Science. 171, 1170-1173 (1971).</li><li>Nicholls, J. G., Martin, A. R., Wallace, B. G., Fuchs, P. 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> From Neuron to Brain. , 315-317 (2001).</li><li>Orlov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Das+Magenganglion+des+Flu&#946;krebses,+Ein+Beitrag+zur+vergleichenden+Histologis+des+sympathischen+Nervensystem.">Das Magenganglion des Flu&#946;krebses, Ein Beitrag zur vergleichenden Histologis des sympathischen Nervensystem.</a> Z. Mikrosk. Anat. Forschung. 8, 67-102 (1927).</li><li>Pasztor, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurophysiology+of+respiration+in+decapod+Crustacea:+The+motor+system.">The neurophysiology of respiration in decapod Crustacea: The motor system.</a> Can. J. Zool. 46, 585-596 (1968).</li><li>Schapker, H., Breithaupt, T., Shuranova, Z., Burmistrov, Y., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+and+ventilatory+correlative+measures+in+crayfish+during+environmental+disturbances+and+social+interactions.">Heart rate and ventilatory correlative measures in crayfish during environmental disturbances and social interactions.</a> Comp. Biochem. Physiol., A. 131, 397-407 (2002).</li><li>Shuranova, Z. P., Vekhov, A. V., Burmistrov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behavioral+reactions+of+fresh-water+crayfish+to+sensory+exposures:+the+autonomic+components.">The behavioral reactions of fresh-water crayfish to sensory exposures: the autonomic components.</a> Zh. Vyssh Nerv. Deiat Im I P Pavlova. 43, 1159-1169 (1993).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Wiese, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilatory+activity+in+free+moving+crayfish+is+indicative+of+its+functional+state+and+perceiving+external+stimuli.">Ventilatory activity in free moving crayfish is indicative of its functional state and perceiving external stimuli.</a> The Crustacean Nervous System. , 526-535 (2002).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Strawn, J. R., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+autonomic+nervous+system+in+decapod+crustaceans.">Evidence for an autonomic nervous system in decapod crustaceans.</a> Inter. J. Zool. Res. 2, 242-283 (2006).</li><li>Wiersma, C. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflexes+and+the+central+nervous+system.">Reflexes and the central nervous system.</a> The physiology of Crustacea, vol II, Sense organs, integration, and behavior. , 241-279 (1961).</li><li>Wilkens, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+control+of+respiration+in+decapod+Crustacea.+Fed.">Neuronal control of respiration in decapod Crustacea. Fed.</a> Fed. Proc. 35, 2000-2006 (1976).</li><li>Wilkens, J. L., Wilkens, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+control+of+cardiac+and+scaphognathite+pacemakers+in+the+crab+Cancer+magister.">Central control of cardiac and scaphognathite pacemakers in the crab Cancer magister.</a> J. Comp. Physiol. 90, 89-104 (1974).</li><li>Wilkens, J. L. <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+cardiac+rhythmicity+and+of+blood+distribution+in+crustaceans.">The control of cardiac rhythmicity and of blood distribution in crustaceans.</a> Comp. Biochem. Physiol. A. 124, 531-538 (1999).</li><li>Yamagishi, H., Ando, H., Makioka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+heartbeat+in+the+primitive+crustacean+Triops+longicaudatus.">Myogenic heartbeat in the primitive crustacean Triops longicaudatus.</a> Biol. Bull. 193, 350-358 (1997).</li><li>Yazawa, T., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+repetitive+cardiac+slowdown+in+the+freely+moving+spiny+lobster,+Panulirus+japonicas.">Spontaneous and repetitive cardiac slowdown in the freely moving spiny lobster, Panulirus japonicas.</a> J. Comp. Physiol. 187, 817-824 (2001).</li><li>Zavarzin, A. A., Zavarzin, A. A., Izbrannye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ocherki po evol utsionnoj gistologii nervnoj sistemy (Essays on the evolutionary histology of the nervous system). III, (1950).</li></ol>

<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>	
<tr>
<td>Scalpel (Stainless steel surgical blades No. 11 for No. 3 handle)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Stainless steel wire (diameter 0.005 inches with the coating 0.008 inches; A-M Systems, Carlsburg, WA)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Impedance detector (UFI, model 2991)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Plastic tubing (Tygon Tubing ID .0812, OD .1492, Cole-Parmer Instrument Company)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fast-drying glue (cyanoacrylate ester, HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Accelerator (HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Adhesive (Eastman, 5-min drying epoxy)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab via a PowerLab/4SP interface (AD Instruments)</a>.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">PowerLab Chart software version 5.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">Chart software version 3.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tbody>
		</table>

measures of heart and ventilatory rates in freely moving crayfish

공포, 비행 또는 싸움 응답이 임박한 약탈 공격에 따른 환경의 생물에 대한 인식을 조사에 대한 기본적인 생리 기초 역할을합니다. 그것이 무척추 동물은 posses 척추 동물의 동일 자율 신경계를하는지, 증거가 보여주는 알려져 있지 않지만 무척추 동물의 내부 환경과 준비 주어진 자극에 behaviorally 행동하는 생물을 조절하는 교감 같은 답변을했습니다. 또한,이 생리 반응은 feasibly 측정하고이 동물의 내부 상태에 대한 생물 학적 인덱스 역할을 수 있습니다. 생리 반응의 측정은 심장 혈관과 호흡 - 시스템의 중추 신경계 통제 조정의 변화를 통해 직접 내부 및 외부 스트레스에 관련된 수 있습니다. 좀 더 구체적으로, 심장 및 환기 속도를 모니터링하는 것은 스트레스 반응 항상 behaviorally 관찰하지 같지는 조치를 제공합니다. 크레이 피쉬는 녹음의 가능성뿐만 아니라 왕새우의 형태 알려진 풍부한 역사, 1888 년 헉슬리까지 거슬러 올라가는, 그리고 잘 공부 전형적인 행동으로 인해 마음과 ventilatory 속도 측정을위한 좋은 모델 생물입니다.

Watch this Scientific Journal Video about Measures of Heart and Ventilatory Rates in Freely Moving Crayfish at JoVE.com

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

무척추 동물은 척추 동물에 대해 설명한 것과 유사 자율 교감과 같은 응답을 보여줍니다. 심장 - 혈관과 ventilatory 시스템의 조정은 유기체 내부 상태를 평가할 수있는 생물 학적 인덱스의 측정을 위해 수 있습니다.

자유롭게 크레이 피쉬 이동에 마음을 Ventilatory 요금 대책

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

measures-of-heart-and-ventilatory-rates-in-freely-moving-crayfish

Research

JoVE Journal

Biology

15.2K 조회수.  University of Kentucky. 무척추 동물은 척추 동물에 대해 설명한 것과 유사 자율 교감과 같은 응답을 보여줍니다. 심장 - 혈관과 ventilatory 시스템의 조정은 유기체 내부 상태를 평가할 수있는 생물 학적 인덱스의 측정을 위해 수 있습니다.

비디오: Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

랫 중심에 엔지니어링된 조직의 주입

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

연구

생물학

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

자유롭게 동물 행동의 신경 회로 활성화의 기록

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

에 잔 심장을 떠올리 Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

크레이 피쉬 오프너 근육의 NMJ에서 역사보기 및 생리학 시범

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

이온화 방사선 다음 γH2AX의 공간적 분포 평가

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA의 붕괴 요금의 측정에<em&gt; 사카로 미세스 세 레비 시아</em사용&gt;<em&gt; rpb1-1</em&gt; 균주

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

대한 유전 및 생화학 적 접근<em&gt; 생체</em&gt; 및<em&gt; 체외</em&gt; 단백질 올리고머의 평가 다음 Ryanodine 수용체 사례 연구

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...