The authors have nothing to disclose.

모든 절차 및 행동 시험은 관리 및 실험 동물의 사용을 위해 동물 관리의 가이드 라인에 대한 캐나다 협의회 준수 프로토콜 번호 06-11-12, 아래 MacEwan 대학의 동물 연구 윤리위원회에 의해 승인되었습니다. 1. 정량 주입 탱크, 솔루션 및 관리 일정을 준비 <ol><li> 동물은 동일한 환경에서 시각적 자극에 시간이나 편향 어떤 혼동을 피하기 위해 하루 중 같은 시간 동안 투여되도록 관리 스케줄을 준비한다. </li><li> 그룹 크기의 수를 필요한만큼 동일, 1.5 L, 명확한 폴리 프로필렌 산란 탱크를 얻습니다. 물고기의 2 그룹에 대한 수 탱크 당 8 물고기의 사용 그룹이 과정에서 나중에 하루에 테스트 할 (3 단계 참조). 하나의 저장 탱크 및 그룹 당 하나의 투여 탱크 (그룹의 2 배 총 수)를 사용합니다. <ol><li> 모든 저장 탱크에 400 μm의 산란 삽입을 놓습니다. F병 서식지 물이나 올바른 온도에서 삼투 물을 역으로 탱크 (제브라 피쉬를 들어, 25 ~ 28 ° C) 온도 생선과 일치 일반적으로 수용된다. 주 : 일부 약물과 버퍼 서식지 물의 화학 성분 사이의 바람직하지 않은 화학적 상호 작용이있을 수 있습니다. 이 상황에서, 역방향 약물 투여없이 또는 최소한의 수족관 염 버퍼링 삼투 물뿐만 아니라위한 대조군을 사용한다. </li><li> 탱크 투여하는 동안 외부의 시각적 자극에 대한 조절 물고기를 방지하기 위해 중립적 인 환경에 있는지 확인하십시오. </li></ol></li><li> 약물 솔루션을 준비합니다. 산란 탱크에 서식지 물과 약물의 적절한 양을 섞는다. 물 1497 ㎖로 높은 등급의 에탄올 (95 % 비 - 변성 에탄올) 3 ㎖를 조합하여 0.2 % 에탄올 용액을 준비한다. 물 1479 ml의 에탄올 21 ml에 결합하여 1.4 % 에탄올 용액을 준비합니다. </li></ol> 2. 그물 물고기와 잇한올 관리 절차 <ol><li> 조심스럽게 그물 서식지 탱크에서 물고기와 산란 삽입을 포함하는 적절한 보관 탱크에 전송. 이상적으로, 모두 그물 제거하기 위해 산란 삽입에 물고기를 집. </li><li> 각각의 물 탱크에있는 모든 물고기와 부드럽게 산란이 유지 탱크에서 삽입하고 적절한 약액 탱크 (그림 1A)에 배치 올립니다. <ol><li> 필요에 따라 투약 시간을 기록한다. 여기에 기재된 절차에 대한 에탄올 용액에 30 분을 사용한다. </li><li> 가능한 경우, 단말기는 정확한 투약 시간을 보장하기 위해 동시에 약액에 모든 실험군의 전달에 도움이있다. 이와 달리, 한 번에 하나의 그룹을 전송하고 각각의 그룹의 투여 시간 (도 1a)을 추적. </li></ol></li><li> 필요한 투여주기의 끝에서, 신중 산란 삽입물을 올려서 에탄올 용액으로부터 물고기를 제거약액 부드럽게 다시 보유 탱크로 배치 아웃. </li><li> 물 탱크에 물고기와 다음 예약 된 투약 시간까지 다시 서식지로 배치, 또는 산란이 그물을 제거하기 위해 다시 저장 탱크에 삽입 배치 조심스럽게 그물. </li><li> 동물 하우징 장비, 집 보유 탱크로 사용되는 탱크와 같은 산란 인서트 동물 매개 변수 내에서 이전에, 가능한 한 바와 같이. 이 관리 절차를 수행하는 동안 모두 그물 제거됩니다. </li></ol> 3. 행동 테스트 <ol><li> 길이 55cm 흰색 방수 바닥 (그림 1B)와 깊이 9.5 cm에서 9.5 cm 폭 빛 / 어둠 무대를 얻습니다. 경기장 흰색에 덮여와 반 검은 색으로 덮여의 절반, 벨크로를 사용하여 경기장의 내부 벽에 흰색과 검은 색 방수 비 반사 종이를 부착합니다. 25 ~ 28 ° C의 온도에서 서식지 물 5cm의 깊이로 무대를 채 웁니다. MAINT테스트를 통해 온도를 줄 알아. </li><li> 경기장에 위치해 위해 흰색 3면 인클로저를 구성하여 외부의 시각적 자극을 Minimalize. 시험 영역은 물 표면에 반사가 발생하지 않습니다 오버 헤드 조명을 확산이 아직 충분히 이동 추적 소프트웨어, 또는 게시물에 대한 밝은 확인 비디오 이미지에서 -hoc 설명서를 정량화. </li><li> 인클로저에 경기장을 배치하고 동작 추적 소프트웨어의 기록 및 운동 분석 매개 변수를 설정합니다. 연구 문제에 따라 5 ~ 15 분으로 시험 시간을 설정합니다. 참고 : 여기에, 우리는 5 분을 사용했다. </li><li> 물고기의 그룹이 서식지 탱크의 연구 영역으로 테스트 할 전송 및 경기장 인클로저의 외부에 배치합니다. 10 분 동안 물고기를 적응. </li><li> / 암 분야의 중심에 적절한 그룹과 장소에서 부드럽게 그물 물고기가 AVO하는 경기장의 장축에 평행하게 위치 할 때 어류를 해제해야 되밝거나 어두운 영역에 물고기를 바이어스 ID입니다. </li><li> 동물이 출시 직후 기록 동작을 시작합니다. 물고기를 추적하거나 점프 물고기 또는 동결에 대한 소프트웨어 문제에 대한 조심. 대상자의 절반 인해 경기장의 끝이 인클로저의 오픈 엔드를 지향하는로 인한 편견에 어떤 혼동을 방지하기 위해 테스트 한 후 무대를 180도 회전합니다. </li><li> 재판은, 부드럽게 그물 종료 및 저장 탱크 또는 서식지 탱크 경기장에서 물고기를 제거 후. </li></ol> 4. 분석 <ol><li> 어두운 영역에 비해 빛에 소요되는 시간을 검사합니다. 각 그룹 및 각 물고기의 경우, 빛과 어두운 영역에서 소비 된 상대 시간을 획득하고 하나의 샘플 T는 - 테스트를 사용하여 분석 (또는 윌 콕슨은 비모수 데이터 signed rank test를, 차이를 총 시험 시간 (하프) 150 초에서) 그룹이 크게 다른 이상 한 지역을 선호하는 경우 확인합니다. </li><li> prefe을 계산, 환경 설정을 비교하려면어두운 영역에서 소요 된 시간에서 광 영역에서 소요과 그룹 간의 차이를 비교하는 시간을 감산함으로써, 보험료 인덱스. T- 테스트는 두 그룹을 비교하는데 사용될 수있다. Tukey의 HSD 포스트 특별 시험을 활용하여 분산의 한 방법으로 분석을 여러 그룹을 비교하여 필요한 경우 (던의 다중 비교 비모수 데이터에 대한 사후 테스트 또는 크루스 칼 - 월리스 테스트). </li><li> 그룹간에 속도, 사행 영역 전환, 수, 부동 비교. Tukey의 HSD 포스트 혹 시험을 이용 일원 변량 분석을 사용하여 필요한 경우 (던의 다중 비교 비모수 데이터에 대한 사후 시험 또는 크루스 칼 - 월리스 시험). </li></ol>

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저자는 그림 1에 사용되는 그의 사진 장비의 사용 여호수아 갤럽을 인정합니다.이 작품은 (TJH에) 자연 과학 및 공학 연구위원회 (NSERC) 캐나다 디스커버리 보조금에 의해 지원되었다.

제브라 피쉬에서 약물 투여와 관련된 이전의 연구는 단순히 약물 솔루션 (12, 16)에 자신의 집 탱크에서 그들을 수송하는 물고기를 그물에 의존했다. 그물은 항상 일치하지 않습니다 종종 인해 상당한 약간의 차이가있는 제브라 피쉬의 탈출 응답하는 데 예상보다 시간이 오래 걸립니다. 기존의 전송 방법은, 유용하지만, 물고기가 물 이외의 총 소요 시간의 양을 감소뿐만 아니라 동물 간의 ...

제브라 피쉬 (다니오 레 리오 (rerio)는) 행동 1 및 의료 연구 2,3를위한 유용한 모델 생물 인도에서 발생하는 작은 경골 종입니다. 제브라 피쉬는 또한 일반적으로 거동에 미치는 영향을 특성화하기 위해 다양한 약리 물질의 시험에 사용된다. 다양한 투여 량 및 약물 투여 스케줄 같은 자극제 4, 5 및 항불안제 에탄올 6-8과 같은 화합물의 투여 후 지브라 피쉬의 거동을 조사하기 위해 사용되어왔다. 우리 연구소는 또한 일반적으로 scototaxic 분석이라 잘 검증 빛 / 어둠 분석 9, 20,에 제브라 피쉬의 불안과 운동에 에탄올 관리의 일정을 다른의 효과를 조사했다. 에탄올 투여 새로운 방법은 장기간 (이십일일) 6 위에 반복적 인 매일 투여 효율성을 높이기 위해 개발되었다 </s&gt;까지. 이전에 사용 된 방법은 그러나, 우리는 관련 시간 비용, 그물 감소 방법을 개발하고자, 물고기 많은 관심 약물의 동시 투여를 정확하게 타이밍 허용 실용적이었다. 기존의 연구를 사용하여 에탄올, 제브라 피쉬는 그물로하고 에탄올과 물을 10 ~ 12의 적절한 혼합물을 포함하는 다른 하나의 탱크에서 전송. 이 방법은 널리 알려진 방법이지만, 제브라 피쉬 그물 것은 도입 약액으로부터 물고기를 제거하는 데 걸리는 시간에 변동을 증가시킬 수있다. 따라서 관심의 화합물에 대한 정확한 노출을 반복 투여를 포함하는 실험의 과정을 통해 달라질 수 있습니다. 전송 시대에 변화에 따른 오류의 원인을 감소시키는 방법은, 따라서 바람직하다. 우리의 방법으로 우리는 각각의 물고기 동일 투여 시간의 결과로, 동시에 모든 물고기를 이동 할 수 있습니다. (여기에 설명 된)을 에탄올 노출 후, 제브라 피쉬 어떤 접점에서 시험 될 수있다불안을 평가하는 것을 포함하여 행동 분석의 R. 새로운 방법을 사용하여 물고기의 그룹을 투여하는 것은 정확하게 복제 및 물고기의 그룹 주제 사이에 걸쳐 투여를 표준화 할 수있는 기능을 넘어 실제적인 용도를 가지고있다. 연구자들은 실험에서 반복 가능성과 정확성을 보장하기 위해 우리의 방법을 이용하여 볼 수 있습니다 한 번에 여러 물고기의 추적을 허용 새로운 소프트웨어의 등장. 생물 심리학위한 모델 생물로서 지브라 피쉬의 광범위한 사용을 고려하면,이 방법은 향후 약리 시험에서의 효율과 실용성을 증가시킬 것이다. 본 패러다임에서, 반복 투여 일정은 대략 인간 마시는 일정을 반영 것을 사용 하였다. 제어, 매일 중등도, 또는 매주-파티 : 생선은 무작위로 1 ~ 3의 그룹에 할당되었다. 투약 일정은 크게 기존의 연구 7 노출 시간을 초과했기 때문에 선택, 기간 21 일이었다. 제어 물고기는 제로 ALCO을 받았습니다HOL, 매일 적당한 물고기는 하루에 한 번 0.2 %의 알코올을 받고, 주간 - 파티 물고기는 일주일에 1.4 %의 알코올을 한 번 받았다. 빛 / 어둠 작업은 철수 2 일 후에 불안을 평가하기 위해 사용되었다. 이것은 한쪽 벽이 백색과 다른쪽에 9 어둡게되는 직사각형 경기장을 사용하는 관리하는 비교적 간단한 테스트이다. 성인 제브라 피쉬는 견고하게 제어 조건 6,9,13에서 경기장의 어두운면을 선호한다. 증가 불안은 작동에 어두운 영역에서 보낸 훨씬 더 많은 시간으로 정의하고, 물고기가 빛 영역에서 소비 상대적으로 더 많은 시간을 소비 할 때 불안을 가정 할 수 감소. 모션 추적 소프트웨어를 다른 정보 변수는 평균 속도, 부동, 사행 포함, 정량화 및 영역 (14)을 전환 할 수 있습니다. 우리 실험실에서 개발 된 투여 방법은 수용성 화합물은 하나 이상의 zebrafi에게 투여되는 임의의 연구에 적용 할 수있다SH. 이 방법론 혜택을 누릴 수있는 많은 다른 약리 에이전트는 현재 제브라 피쉬에서 테스트되고있다. 일반적으로 시험 화합물을 에탄올과 유사한 방식에 용해 니코틴 chlordiazepoxide, 부스 피론 및 스코 폴라 민을 포함한다; 물에 화학 물질의 적절한 양을 혼합함으로써. 따라서,이 절차의 일반적인 범위는 훨씬 더 넓은 및 에탄올에 제한되지 않는다. 또한, 여러 일에 대한 약물 투여 후, 빛 / 어둠 작업이 이용 될 수있는 많은 행동 검사 중 하나입니다. 약물 투여 후 또는 철회 동안 이용 될 수있는 다른 인기있는 분석은 새로운 탱크 다이빙 테스트 (15) 및 (16) 천수와 같은 사회적 행동의 테스트를 포함한다. 다음 절차를 반복 관심 약리 화합물을 함유 한 용액에 물고기 또는 물고기 개별 그룹을 전송하는 효율적인 방법을 개설 할 것이다. 또한, 빛 / 어둠 테스트와 시험 불안의 과정알코올 투여 긴 스케줄에 노출 된 후 철수에 물고기의 그룹으로 설명한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Three Shelf benchtop housing system</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/research-systems/z-hab-system/">http://aquatichabitats.com/research-systems/z-hab-system/</a></td>
		</tr>
		<tr>
			<td>1.5L Spawning tank w/400 micron baffle</td>
			<td>Aquatic Habitats</td>
			<td>N/A</td>
			<td><a href="http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/">http://aquatichabitats.com/consumables/tanks-lids-and-baffles/info/nursery-tank-and-baffles/</a></td>
		</tr>
		<tr>
			<td>Pure Grain Ethanol</td>
			<td>Luxco, INC</td>
			<td>N/A</td>
			<td><a href="http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&amp;aid=3759149&amp;store=Scientific">http://www.luxco.com/public/brands/brands.asp?brandid=21</a></td>
		</tr>
		<tr>
			<td>Ethovision XT Motion tracking software</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td><a href="http://www.noldus.com/animal-behavior-research/products/ethovision-xt">http://www.noldus.com/animal-behavior-research/products/ethovision-xt</a></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf Canada</td>
			<td></td>
			<td><a href="http://www.eppendorf.ca/int/index.php?l=211&amp;pb=e396f9e705e754a3&amp;action=products&amp;contentid=1&amp;catalognode=9477.">http://www.eppendorf.ca/int/index.php?l=211&#38;pb=e396f9e705e754a3&#38;action=products&#38;contentid=1&#38;catalognode=9477.&#160;</a></td>
		</tr>
		<tr>
			<td>Light/Dark Arena</td>
			<td>Custom</td>
			<td></td>
			<td>Construct as per procedure description. 9.5cm wide, 9.5cm deep, 55cm long</td>
		</tr>
	</tbody>
</table>

a novel method of drug administration to multiple zebrafish (danio rerio) and the quantification of withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

상기 한 바와 같이이 일관되고 정확하게 시간에 에탄올 관리의 지속 시간 중요하다 제브라 피쉬와 약리 연구의 정확성과 제어를 유지합니다. 우리의 절차는 절차 투여의 용이성 및 처리량을 증가시킬 수있다. 주간-파티 또는 매일 중등도 중 일정에 에탄올을 투여 대조군과 비교, 빛 / 어둠 테스트로 측정 변경된 불안 수준, 결과. 이틀 마지막 투여 후 시험 할 때, (어떤 술을받은,하지만 여전히 투?...

Watch this Scientific Journal Video about A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal at JoVE.com

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

여러 제브라 피쉬에의 약국 (FDA)의 새로운 방법 (<em&gt; 다니오의 레 리오 (rerio)</em&gt;)와 철수의 정량화

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

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Research

JoVE Journal

Neuroscience

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

12.0K 조회수.  MacEwan University. A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

비디오: A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

연구

신경과학

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

쥐에서 의약품 자체 관리하는 동안 초음파 Vocalizations 평가

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

가스 크로마토 그래피에 의한 operant 에탄올 자기 관리 및 에탄올 결정시 에탄올의 Microdialysis

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Zebrafish의 성인 Telencephalon의 자상 부상 : 척추 동물의 뇌 신경 발생과 재생을 조사하는 방법

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

소뇌 과립 신경 세포 배양에서 Fluorescein Diacetate-Propidium Iodide 이중 염색을 이용한 신경 생존력의 평가

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

만성 외상성 뇌증 모델링을위한 새로운 방법<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

살아있는 세포에 Sod1 돌연변이 단백질 집계 형성의 높은 콘텐츠 정량화에 대 한 분석 결과 개발

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

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

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (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 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 ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

시각화 및 약리 대상의 특성에 대 한 간단 하 고 강력한 방법으로 autoradiography

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...