이 작품은 베이징의 자연 과학 재단 (No.7162124), 그리고 중국 의학의 베이징 대학에 대 한 티베트 ao 재단에 의해 지원 되었다.

모든 동물 지침에 따라 중국 입법에 윤리적인 사용 및 실험실 동물 들에 의해 유지 되었다.
1. 사전 모델링 준비
<ol><li>
실험실 준비
	<ol>
<li>실험 전에 적어도 30 분 동안 UV 램프를 실행 합니다.</li>
		<li>실험실 온도 25 ± 3 ° C, 상대 습도 30%의 주위에 통제 한다.</li>
		<li>6:00 빛 연구소에 돌아서 18:00 12 h/12 h 명암 주기 설정 하에 그것을 해제.</li>
	</ol>
</li>
	<li>
여러 플랫폼 상자 건설
	<ol>
<li>110 × 60 × 40 cm3의 표지 없이 불투명 한 플라스틱 탱크를 생성 합니다.</li>
		<li>15 원형 플랫폼 수정 (h = 8 m, d = 6.5 c m)는 탱크의 바닥에 어떤 질서에 세 개의 행과 다섯 개의 열 배포. 열과 행 13 cm 10 cm이 약 각 플랫폼 사이 두고 충분 한 공간.</li>
		<li>탱크의 측면 쪽에 물 출구를 설정 하 고 수도 꼭지를 설치.</li>
		<li>그것에 걸려 음식 상자와 탱크에 대 한 철 철사 그물 세공 커버를 확인 합니다.</li>
	</ol>
</li>
	<li>
그룹화 및 주택 쥐 참고: 8 주 정도, 무게 약 200-210 g의 Wistar 남성 쥐 실험에 사용 됩니다. 쥐는 모델링 과정 그룹에 살고 있습니다.<ol>
<li>마커 펜으로 쥐의 꼬리 뿌리를 번호.</li>
		<li>쥐 무게, 매우 가벼운 또는 무거운 것 들을 제외 하 고 모델 및 제어 그룹으로 나머지를 임의로 분할.</li>
		<li>부드럽게 깨끗 한 연습장에 쥐를 넣어 하 고 적어도 3 일 동안 연구실에 적응 하도록 허용. 충분 한 물과 식량 공급을 제공 합니다.</li>
	</ol>
</li>
</ol>2입니다. 모델링 MMPM
참고: 과정 18시 시작 하 고 8 시 다음 날, 21 일 이상, 하루 14 h의 총에 끝납니다. 방해 요소를 방지 하려면 동일한 사람이 동일한 실험실 외 투를 입고 있는 동안 전체 실험을 수행 하는 데 필요한입니다. 10 Wistar 쥐 실험에 사용 됩니다.
<ol><li>평평한 표면, 예를 들면, 바닥에 탱크를 배치 합니다. 다음 따뜻한 (25 ± 3 ° C) 물, 플랫 플랫폼 아래 약 1 ㎝의 약 7 cm로 탱크를 채우십시오.</li>
	<li>1 일에 대 한 탱크에 있는 모든 쥐에 대 한 충분 한 음식과 음료를 준비 합니다. 사료와 물을 음식 상자에 넣고 덮개에 걸어. 참고: 일부 스마트 쥐 음식 상자에 배워야 했다. 만약 그렇다면, 탱크에 다시 그들을 드라이브.</li>
	<li>감 금 소에서 모델 그룹 쥐를가지고, 그들을 잡아, 꼬리 그리고 탱크에 부드럽게 넣어. 그들의 공포 물의 동기를 플랫폼 대신 물에 모든 쥐를 시작 합니다. 모든 쥐 쥐 제어 그룹의 충분 한 음식과 물을 함께 원래 그들의 감 금 소에 체류 하는 동안에, 서 서 플랫폼을 얻을 다는 것을 확인 하십시오.</li>
	<li>탱크 커버. 실수로 부상을 피하기 위해 쥐를 모니터링 합니다. 쥐 플랫폼에 등반 하지 않고 1 시간 이상 물에 유지, 탱크에서 그것을 선택 하 고 테스트에서 그것을 제거.</li>
	<li>14 h, 후 모델 쥐 탱크 중 고 건조 기를 가진 그들의 머리를 건조. 그것은 퇴색 하는 경우 쥐의 꼬리를 다시 표시 합니다. 그들의 원래 감 금 소에는 쥐를 반환 하 고 충분 한 음식과 물을 함께 그들을 제공.</li>
	<li>탱크의 구석구석을 플러시. 탱크의 한쪽을 상승 하 고는 하 수를 유출 하는 수도 꼭지를 엽니다.</li>
	<li>75% 에탄올 스프레이 탱크를 소독 하 고 UV 빛에 노출.</li>
</ol>3. 모델: 행동 테스트
참고: 모든 테스트 행동 실험실에서 수행 됩니다. 잡음과 여분의 빛 허용 되지 않습니다 테스트 하는 동안 방해를 피하기 위해. 가능 하면 같은 사람을 사용 하 여 각 테스트를 실시. 어두운 코트와 장갑은 그레이 스케일 이미지 처리에 인식 필요 합니다. 자주 수행 먼저 쥐 행동에 최소 영향.
<ol><li>
자주
	<ol>
<li>워크스테이션에 올바르게 연결 되어 있고 상자의 모든 코너 커버 확인을 열기 필드 상자에 레코더를 확인 합니다. 상자에서 그림자를 제거 하기 위해 조명을 조정 합니다.</li>
		<li>그들의 원래 장에 행동 실험실에 쥐를 이동 합니다. 그들이 테스트를 하기 전에 적어도 1 시간에 대 한 적응 수 있습니다.</li>
		<li>깨끗 하 고 아무 배설물 또는 냄새 이전 실험에서 왼쪽을 75% 에탄올과 상자를 청소.</li>
		<li>그 뒤로 여 케이지에서 쥐를 제거 하 고 부드럽게 상자의 중앙 영역에 그것을 넣어. 신속 하 게 차단 주사를 하지 상자에서 무기를 후퇴.</li>
		<li>쥐의 숫자를 입력 하 고 녹음을 시작 합니다. 계산 하 고 양육 하 고 등반을 포함 하 여 쥐의 수직 활동의 주파수를 기록 합니다.</li>
		<li>5 분 후 녹음을 중지 하 고 상자에 쥐를 데리고 감 금 소에 그것을 반환 합니다.</li>
		<li>단계 3.1.3-3.1.6 모든 쥐 테스트 완료 될 때까지 반복 합니다.</li>
	</ol>
</li>
	<li>
EPM
	<ol>
<li>자주 (단계 3.1.1 ~ 3.1.2)를 사전 확인 하 고 새 환경 순응 단계를 수행 합니다.</li>
		<li>그것의 뒤에 의해 감 금에서 쥐를 제거 하 고 부드럽게 두 팔의 접합 부분에 그것을 넣어. 왼쪽된 오픈 팔 쪽으로 쥐 고 신속 하 게 두고 총을 차단할 수 없습니다.</li>
		<li>쥐의 숫자를 입력 하 고 녹음을 시작 합니다. 계산 하 고 다른 팔 입구의 주파수를 기록 합니다. 쥐 테스트에 미로에서 떨어지면, 그것을 데리 고 미로 다시 보낼. 데이터 분석에 대 한 자세한 정보를 기록 합니다.</li>
		<li>5 분 후 녹음을 중지 하 고, 쥐를가지고 한 감 금 소를 반환 합니다.</li>
		<li>배설물을 제거 하 고 75% 에탄올 전 쥐의 냄새를 제거 하는 미로 닦아냅니다.</li>
		<li>단계 3.2.2-3.2.5 모든 쥐 테스트 완료 될 때까지 반복 합니다.</li>
	</ol>
</li>
	<li>
ES 시험
	<ol>
<li>80 cm (25 ± 3 ° C) 따뜻한 물로 수영 탱크 (70 × 30 × 110 c m3)를 채우십시오. 참고: 경우 탱크에는 온도, 물 온도 설정 되어야 합니다 약 37 ° C, 쥐의 체온에 비슷합니다. 그렇지 않으면, 그것을 일정 하 게 유지 하는 실내 온도에 그것을 설정 합니다.</li>
		<li>핀 각 쥐 움 큼에 대 한 부하를 만들고 그것의 꼬리에 그것을 부드럽게 넥타이. 부하 쥐의 체중의 10% 무게.</li>
		<li>꼬리는 쥐를 잡아 고 수영 탱크에 그것을 던져. 쥐 모이 또는 벽에 집착 하는 경우 떨어져 그들을 설정 하 고 다시 물으로 그들을 드라이브.</li>
		<li>타이밍을 시작 쥐 중지 타이밍 모두 소진 되 면 물에 넣으면 순간에는 입과 코 이상 10 물 아래와 물 투쟁 실패로 설명 된다 s. 참고: 때때로, 피로 익사 발생 갑자기. 기록 하 고 동시에 동물을 저장할 충분 한 경험을가지고 해야 합니다.</li>
		<li>다른 사람을 방해 하지 않고 물 소진된 쥐를 제거 합니다. 그들의 머리를 건조 하 고 다시 그들의 숫자를 표시 한 케이지로 다시 보내.</li>
		<li>한 그룹 완료 후 탱크에 물의 변경 합니다. 모든 쥐, 완료 후 수영 탱크, 빈 하 고 깨끗 하 고 에탄올 및 자외선 소독.</li>
	</ol>
</li>
</ol>4. 모델 평가: 중앙 신경 전달 물질 검출
<ol><li>그것은 의식이 될 때까지 10 %chloral 하이드 레이트 (3 mL/kg)의 복 막 내 주입으로 쥐를 anesthetize.</li>
	<li>쥐 목을 벨.</li>
	<li>-중간 라인을 따라 절 개를 경도, 양쪽 모두에 두개골을 열고 고 뇌를 노출 시킵니다. 두개골을 넘겨, 두뇌, 제거 하 고 얼음 가방에 두뇌를 넣어.</li>
	<li>하 고 주변 조직으로 명확한 경계는 두뇌의 중앙 부분에 다이아몬드 모양 지역 이다 시상 하 부를 제거 합니다. 무 균 튜브에 그것을 배치 하 고 액체 질소 동결. -80 ° C 냉장고에서 모든 샘플을 저장 합니다.</li>
	<li>고성능 액체 크로마토그래피 (HPLC)8를 사용 하 여 시상 하 부에 다와 5-HT의 콘텐츠를 검색.</li>
</ol>

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X., 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=Distinct+behavioral+and+brain+changes+after+different+durations+of+the+modified+multiple+platform+method+on+rats:+An+animal+model+of+central+fatigue.">Distinct behavioral and brain changes after different durations of the modified multiple platform method on rats: An animal model of central fatigue.</a> PloS One. 12 (5), e0176850 (2017).</li><li>Tang, X., Yang, L., Sanford, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+variation+in+sleep+and+motor+activity+in+rats.">Individual variation in sleep and motor activity in rats.</a> Behavioural Brain Research. 180 (1), 62-68 (2007).</li><li>Stanford, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Open+Field+Test:+reinventing+the+wheel.">The Open Field Test: reinventing the wheel.</a> Journal of Psychopharmacology. 21 (2), 134-135 (2007).</li><li>Ahn, S. 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=Basal+anxiety+during+an+open+field+test+is+correlated+with+individual+differences+in+contextually+conditioned+fear+in+mice.">Basal anxiety during an open field test is correlated with individual differences in contextually conditioned fear in mice.</a> Animal Cells and Systems. 17 (3), 154 (2013).</li><li>Costa, A. A., Morato, S., Roque, A. C., Tin &#243;s, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+model+for+exploratory+activity+of+rats+with+different+anxiety+levels+in+elevated+plus-maze.">A computational model for exploratory activity of rats with different anxiety levels in elevated plus-maze.</a> Journal of Neuroscience Methods. 236, 44-50 (2014).</li><li>Liu, Z., Wu, Y., Liu, T., Li, R., Xie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+regulation+in+a+rat+model+of+exercise-induced+chronic+fatigue.">Serotonin regulation in a rat model of exercise-induced chronic fatigue.</a> Neuroscience. 349, 27-34 (2017).</li><li>Foley, T. E., Fleshner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroplasticity+of+dopamine+circuits+after+exercise:+implications+for+central+fatigue.">Neuroplasticity of dopamine circuits after exercise: implications for central fatigue.</a> NeuroMolecular Medicine. 10 (2), 67-80 (2008).</li><li>Leite, L. H., Rodrigues, A. G., Soares, D. D., Marubayashi, U., Coimbra, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+fatigue+induced+by+losartan+involves+brain+serotonin+and+dopamine+content.">Central fatigue induced by losartan involves brain serotonin and dopamine content.</a> Medicine &amp; Science in Sports &amp; Exercise. 42 (8), 1469-1476 (2010).</li></ol>

저자는 공개 없다.

MMPM 원래 수 면 박탈9에 대 한 설계 되었습니다. 쥐 플랫폼 바닥에 고정 물 탱크에 착수 한다. 물의 본능적 인 두려움에 의해 구동, 쥐 플랫폼에 서 서 유지 하 고 아니 수 면 발생 합니다. 연구는 수 면 부족의 다른 시간 쥐 행동 및 인식 장애10, 부정적인 감정을11및 중앙 피로 포함 하 여, 분위기에 다양 한 변화를 이끌어 보여줍니다. 일부 연구 ?...

피로1인간의 건강 위협 하는 주요 요인 중 하나 이다. 지난 수십 년간에서 다양 한 연구는 피로 주변 실행 하지만 중앙 기반, 그리고 항상 감정과 인지 장애와 함께 입증 해야 합니다. 이탈리아 생리학자 A. Mosso 먼저 단어 중앙 피로2제안. 그것은 제한 된 자발적인 활동 및 중앙 신경 조직 (CNS)3의 충 동 장애 때문 인지 장애로 일반적으로 정의 된다. 주변 근육 피로와 비교해, 중앙 피로 CNS, 필연적인 감정/행동 장애, 우울증, 불안, 인지 장애, 그리고 기억 상실을 포함 하 여에 있는 변화를 강조 합니다. 한 연구는 많은 요인은 과도 한 신체 활동 및 정신적인 스트레스는 매우 필수적인4중앙 피로 일으킬 수 보여줍니다. 병 인에 관해서는 트립토판 kynurenine 통로 가설5 같은 이론 설명; 특정 경로에 변화 그러나, 더 깊이 있는 연구는 여전히 중앙 피로의 중앙 주변 상관 관계를 공개 해야 합니다.
중앙 피로의 기본 메커니즘은 아직 불분명로 효과적인 동물 모델 추가 연구에 대 한 확실히 중요 하다. 기존 피로 모델은 주로 러닝6 과 체중 로드 수영7, 정신적 요인에 작은 우려와 같은 과도 한 운동에 의해 유발 됩니다. 더 나은 중앙 피로의 개발 시뮬레이션, 우리의 그룹은 MMPM 쥐 모델 개발. 모델링 하는 동안 쥐의 수 면 시간 부분을 포함 하 여 긴 시간에 대 한 다중 플랫폼 상자에서 좁은 플랫폼에 서 서 남아 있습니다. 과도 한 운동 모델에서 다른 MMPM 모델 중앙 피로의 복잡 한 병 인을 고려 하 여 정신적인 요인으로 부분 수 면 부족을 사용합니다.
모델 평가 대 한 우리는 불안 분위기와 자발적인 활동을 결정 하는 자주 및 EPM 테스트를 사용 합니다. ES 시험 주변 근육 성능을 측정 하기 위해 수행 됩니다. 또한, 우리 쥐의 뇌를가지고 고 도파민 (DA) 감지 / 중앙 신경 전달 물질의 차이 관찰 하 두 hypothalamuses에서 세로토닌 (5-HT) 콘텐츠.
프로토콜 아래는 모델 중앙 피로 반복된 신체 활동 및 수 면, 인간의 인생에서 일반적인 상태를 흉내 낸의 부족에 의해 유도 된 설계 되었습니다. 그러나, 모델 기간을 조정 하 여 그것은 사용할 수 있습니다 다른 많은 분야에서 처럼 수 면 관측 및 스트레스 연구에서. 미래에 연구, 우리이 모델 더 CNS 변화와 중앙 피로의 pathogenesis 메커니즘을 주변 시스템과 그들의 연결을 발견 도움이 될 것입니다 바랍니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>multiple platform sleep deprivation water tank</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>110cm x 60cm x 40cm. There are 15 plastic small platforms at the bottom. The small platform is 6.5cm in diameter and 8cm high</td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Beijing Weitong Lihua Experimental Animal Technology Company</td>
			<td>license number SYXK (Beijing) 2016-0011</td>
			<td>Use 32 Wistar healthy male rats ,8 week old (200-210 g)</td>
		</tr>
		<tr>
			<td>Agilent 1100LC high performance liquid chromatograph&#160;</td>
			<td>Agilent</td>
			<td>&#160;G1379A, G1311A, G1313A , G1316A&#160;</td>
			<td>&#160;G1379A, G1311A type chromatographic pump, G1313A automatic sampler, G1316A column temperature box</td>
		</tr>
		<tr>
			<td>DECADE II SDC electrochemical detector</td>
			<td>Dutch ANTEC company</td>
			<td></td>
			<td>glassy carbon electrode, Ag/AgCl reference electrode, workstations (Clarity CHS)</td>
		</tr>
		<tr>
			<td>Biofuge Stratos high-speed refrigeration centrifuge</td>
			<td>HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VCX130 ultrasonic fracturing instrument</td>
			<td>SONICS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACS-ZEAS electronic scale</td>
			<td>Phos technology development, Beijing.</td>
			<td></td>
			<td>The weight of the weighing rats can be accurate to 0.1g.</td>
		</tr>
		<tr>
			<td>Open Field Box</td>
			<td>Customization,it is provided by the neuroimmunological laboratory of Beijing University of Chinese Medicine</td>
			<td></td>
			<td>wooden box of open field&#160; 100 cm by 100 cm x 40 cm, inside wall and bottom as the gray.The bottom is divided into 25 equal area squares, each of which is 20cm x 20cm, and the 16 grids along the outer wall are the external ones, and the other 9 grids are central.The camera is mounted above the median.</td>
		</tr>
		<tr>
			<td>Elevated Plus-maze</td>
			<td>Beijing zhongshi dechuang technology development co. LTD.</td>
			<td></td>
			<td>The open arms and close&#160; arms of the cross are composed of 30cm x 5cm x 15cm, and the central area is 5cm x 5cm, with a camera mounted above the center and 45cm high.</td>
		</tr>
		<tr>
			<td>rat swimming bucket.</td>
			<td>Zhenhua biological instrument equipment co., LTD. Anhui,China.</td>
			<td></td>
			<td>The volume of plastic drum is 70cm x 30cm x 110cm, which is used for swimming in rats.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Shiya instrument co., LTD., changzhou,China.</td>
			<td></td>
			<td>Control water temperature</td>
		</tr>
		<tr>
			<td>Small water pump</td>
			<td>Xincheng technology co., LTD., chengdu,China.</td>
			<td></td>
			<td>Used for water tank and swimming behavior.</td>
		</tr>
		<tr>
			<td>Ethovition3.0 behavioral software.</td>
			<td>Nuldus,Netherlands</td>
			<td></td>
			<td>Measurement analysis of rat behavior videos.</td>
		</tr>
	</tbody>
</table>

a rat model of central fatigue using a modified multiple platform method

이 문서에서는, 우리는 수정 사용 하 여 중앙 피로의 쥐 모델을 도입 여러 플랫폼 메서드 (MMPM). 여러 플랫폼 상자는 물 탱크 바닥에 좁은 플랫폼으로 설계 되었습니다. 모델 쥐 탱크에 투입 되었고 빈 제어 그룹 대비 설정 연속 된 21 일 동안 하루 14 h (18시-8:00)에 대 한 플랫폼에 서 서. 모델링의 끝에, 모델 그룹에서 쥐 명백한 피로 모습을 보여주었다. 모델을 평가 하기 위해 여러 가지 행동 테스트를 수행 했습니다, 오픈 필드를 포함 하 여 테스트 (자주)는 높은 플러스 미로 (EPM) 테스트, 그리고 철저 한 수영 (ES) 테스트. 결과 그 불안, 공간 인식 장애, 불 쌍 한 근육 성능과 거부 자발적인 활동 모델 쥐에 중앙 피로의 진단을 확인 보여주었다. 중앙 신경 전달 물질의 변화는 또한 결과 확인합니다. 결론적으로, 모델은 성공적으로 중앙 피로, 시뮬레이션 및 모델 미래 연구는 질병의 병 적인 메커니즘을 공개 도움이 될 수 있습니다.

우리는 MMPM를 사용 하 여 중앙 피로의 쥐 모델을 설명 합니다. 24 Wistar 쥐 임의로 제어 그룹 및 각 그룹에 12 쥐 모델 그룹으로 나누어집니다. 모델 장치는 물 탱크 하단 (그림 1)에 좁은 플랫폼으로 설계 되었습니다. 모델 쥐 21 일 (그림 2)에 대 한 부분적인 수 면 시간을 포함 하 여 하루 14 h에 대 한 플랫폼에 서 서.
<p class="jove_co...

Watch this Scientific Journal Video about A Rat Model of Central Fatigue Using a Modified Multiple Platform Method at JoVE.com

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

여기, 선물이 수정 사용 하 여 중앙 피로의 쥐 모델을 소개 하는 프로토콜 여러 플랫폼 메서드 (MMPM).

쥐 모델에 수정 사용 하 여 중앙 피로의 여러 플랫폼 메서드

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

a-rat-model-central-fatigue-using-modified-multiple-platform

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.9K 조회수.  Beijing University of Chinese Medicine. 여기, 선물이 수정 사용 하 여 중앙 피로의 쥐 모델을 소개 하는 프로토콜 여러 플랫폼 메서드 (MMPM).

비디오: A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

동적 클램프 기법을 사용 랫 Midbrain Dopaminergic 뉴런에서 NMDA 수용체의 전도의 응용

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

연구

신경과학

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

위치 선택성과 유전자는 수정 된 유전자 총을 사용의 Organotypic 뇌 조각에 타겟팅

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

스프 라그 - 돌리 쥐에 Nodose 신경절 주입하는 방법

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

녹화 및 자극 애플리케이션을위한 높은 접촉 밀도의 제작, 평면 인터페이스 신경 전극

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

동적 정량적 감각 테스트는 중앙 통증 처리의 특성을

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

다발성 경화증의 마우스 모델에서 진보적 인 신경 학적 장애를 측정

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

거미 막 밑 출혈의 Murine 모델에 대뇌 Vasospasm의 정량화에 대 한 체적 메서드

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

여러 중추 신경계 영역에서 쥐 아교 세포의 해부 및 분리

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

쥐 뇌 조직을 위한 소수성 조직 청산 방법

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...