저자는 훌륭한 물고기 사육 및 기술 지원 시샤 루오에게 감사의 말씀을 전합니다. 이 작품은 건강 보조금 R21EY019401 (RT)와 P30EY04068 (RT)의 국립 연구소에 의해 자금 지원, 그리고 웨인 주립 대학 안과학 교실에 실명을 방지하는 연구에서 제한 교부금 등, RT에 기금을 시작했습니다. JT는 웨인 주립 대학 대학원에서 제공하는 토마스 C. 럼블 원정대에 의해 지원되었다.

이 프로토콜에 설명 된 모든 절차는 의학의 웨인 주립 대학 대학원에서 동물 사용위원회에 의해 승인되었습니다. 1. 어두운 적응 <ol><li> 전송 어두운 인클로저에 일반 주택 시스템에서 10 성인 흰둥이 나 색소 물고기를 ~. 가능한 경우, 탱크를 통해 정상적인 물 흐름을 허용 zebrafish의 주택 모듈에 내장되어 어두운 인클로저를 사용합니다. (이러한 시스템을 사용할 수없는 경우, 산소와 생선을 공기에 쐬다을 확인하고, 완전히 어두운 인클로저에 물고기 탱크를 배치). </li><li> 10 일 동안 어둠 속에서 물고기를 유지합니다. 동물을 먹이거나 어두운 상자에 새로운 물고기를 추가 할 때, 빛의 긴 기간에 물고기를 노출 피하기 위해 가능한 한 빨리 이동해야합니다. </li></ol> 2. 자외선 노출 <ol><li> UV 소스의 전원이 꺼져 있는지 확인합니다. 형광 입체 현미경에서 UV 빛의 필라멘트를 제거합니다. <ol><li> 반전을 사용UV 필라멘트를위한 대와 같은 15cm 직경의 유리 페트리 접시 (또는 유사한 2cm 높이의 것). 실험실 벤치 페트리 접시 테이프. </li><li> 테이프 UV 라이트 필라멘트 반전 페트리 접시의 상단입니다. 정렬되도록 페트리 접시 필라멘트 돌출​​의 끝 ~ 2mm. </li></ol></li><li> 250 ML 유리 비커를 구합니다. 바닥의​​ ½, 측면, 다시 알루미늄 호일로 비커, 호일의 &quot;반짝&quot;면이 비커의 내부를 직면해야하는 커버. 비커를 졸업 한 경우, 노출 된 비커의 명확한 절반을 남겨 호일로 비이커의 말로 절반을 커버. </li><li> 물고기 설비 시스템 물 100 ㎖와 250 비이커를 입력합니다. </li><li> 4 L 비이커에 250 ML의 비커를 놓습니다. 수위도 250 ML의 비커 100 ML 물 줄 때까지 물 4 L 비이커를 입력합니다. </li><li> 250 비이커에 10 어두운 치료 동물의 최대를 추가합니다. 알루미늄 작은 조각에 250 ML의 비커 커버호일. </li><li> UV 필라멘트 바로 옆에 전체 비커 장치를 놓습니다. 필라멘트 4 L 비커의 외부를 만지고 250 비이커의 노출 부분을 직면해야한다. 250 비이커는 4 L 비커의 바닥 중앙에 있는지 확인합니다. </li><li> 노출되는 동물을 허용하지만, UV 필라멘트의 끝을 보는 모든 실험실 인력을 방지 4L 비커 뒤에 큰, 불투명 한 화면에 배치합니다. 경고 :이 장벽 UV 소스에 전원을 켜기 전에 위치에 있는지 확인하십시오. </li><li> UV 전원을 켭니다. 30 분 타이머를 설정합니다. 어떤 필요에 경고 라벨이 의심 실험실 직원이 실수로 UV 방사선에 자신을 노출하지 않도록 배치합니다. </li><li> 30 분 후, UV 전원을 차단합니다. 물고기와 250 비이커를 제거합니다. 참고 : UV 노출의 여러 라운드가 필요한 경우, 노출 프로토콜을 반복하기 전에 (~ 20 분) 아래로 전원을 식히십시오. t를 교체해야합니다그는 각각의 노출 신선한 시스템 물 물 100ml. 4 L 비이커에 물을 교체 할 필요가 없습니다. </li></ol> 3. 할로겐 램프 빛 노출 <ol><li> 1.8 L 명확한 아크릴 수조로 250 비이커에서 물고기를 전송합니다. 오버 플로우 표시로 탱크를 채우십시오. </li><li> 할로겐 램프 빛 치료 영역을 설정합니다. 철물점에서 네 개의 250 W의 할로겐 유틸리티 램프를 얻습니다. 같은 방향에있는 두 개의 램프를 직면하고, 중앙에 떨어져 29cm를 정렬합니다. 비슷한 방식으로 다른 두 개의 램프를 정렬합니다. <ol><li> 그들은 램프의 두 세트 사이에 ~ 73cm를 떠나, 램프의 첫 번째 집합을 직면되도록 램프의 두 번째 세트를 놓습니다. 이 29 X 73cm의 직사각형 모양의 조명 치료 영역을 만듭니다. </li></ol></li><li> 철물점에서 작은 팬을 구하십시오. 그냥 가벼운 치료 영역의 외부 램프의 두 집합 사이의 등거리 팬을 놓습니다. </li><li> 의 중앙에 물이 가득 두 개의 1.8 L 탱크를 배치램프의 두 집합 사이의 등거리 빛 치료 영역. 램프와 탱크의 외부 벽 사이의 거리 ~ 29cm이어야한다. 참고 : 하나의 1.8 L의 탱크에 10 물고기를 치료하는 경우에도이 배열을 사용합니다. 두 탱크는 적절한 범위 내에서 각 탱크의 물 온도를 유지하기 위해 필요합니다. </li><li> 각 탱크에 산소 통풍 장치를 놓습니다. </li><li> 팬에 가장 가까운 끝 부분에 1 ~ 2 센티미터 간격을두고 명확한 아크릴 뚜껑 각 탱크를 포함한다. 는이 틈새에 공기를 불어되도록 팬을 정렬합니다. 탱크 중 하나 또는 둘 모두에 온도계를 놓습니다. </li><li> 빛, 팬 및 에어 레이터의 전원을 켭니다. 최대 4 일을위한 가벼운 치료를 유지합니다. 이 동물에 더 많은 스트레스 수질과 결과에 영향을 미치기 때문에 물고기, 빛 치료 기간 동안 공급 할 수 없습니다. </li><li> 매일 온도와 수위를 모니터링합니다. 30-33의 온도를 유지 ° C. 필요한 경우, 팬 속도 및 / 또는 탱크와 리 사이의 거리를 조정ghts. <ol><li> 필요에 따라, 일반적으로 일상적인 시스템 물 각 탱크 떨어져 꼭대기. </li><li> 항상 거의 100 %의 생존율을 유지하기 위해 건강한 성인 zebrafish의를 (연령 일반적으로 6-9개월)를 사용합니다. 물고기 탱크에서 시체로 발견되는 경우, 즉시 제거하고 전체 탱크에 물을 교체합니다. </li></ol></li></ol> 4. 조직 컬렉션 <ol><li> 할로겐 빛 치료의 발병 후 48 시간이 치료 영역에서 물고기를 제거합니다. <ol><li> 신선한 에탄올 포름 알데히드 정착액 (1 일부로 37 % 포름 알데히드, 9 부품 100 % 에탄올)을 준비합니다. </li><li> 2 페녹시 에탄올 (0.4 ㎎ / L)의 마취제 과다 복용으로 제브라 피쉬를 안락사. </li><li> 종이 타월로 안락사 제브라 피쉬를 전송합니다. 곡선 집게를 사용하여 눈을 명백히. </li><li> 4 ° C.에 정착하고 저장 O / N에 탈핵의 눈을 배치 </li></ol></li><li> 눈을 Cryoprotect. <ol><li> 실온에서 30 분 동안 5 % 자당 1X PBS에 눈을 씻고, 다음으로 대체2 시간 동안 신선한 5 % 자당 1X PBS. 다음, 4 ℃에서 30 % 자당 1X PBS O / N에 눈을 씻어 4 ° C.에서 조직의 냉동 중간 30 % 자당 1X PBS O / N의 1:1 (등분)로 눈을 씻어 </li></ol></li><li> -80에서 100 % 조직 동결 중간 저장소에 눈을 포함 ° C. 동양의 눈 있도록 조직의 cryosectioning 것은 지느러미 / 복부 축에서 수행됩니다. </li><li> 유리 슬라이드에 동결 절단 (Cryosections) 조직과 장소. 55 ℃에서 2 시간 동안 따뜻한 슬라이드 -80 매장 슬라이드 ° C 또는 즉시 표준 면역을 수행합니다. </li><li> 형광 현미경 40,51와 단면 조직 및 이미지의 표준 면역을 수행합니다. </li></ol>

저자는 공개 아무것도 없어.

여기에서 우리는 광범위한 광수 손실과 강력한 재생 반응의 지속적인 밝은 빛에 노출 결과로 짧은 UV 노출을 결합하는 것을 보여줍니다. 개인 LIRD 방법에 비해이 결합 된 방법은 또한 망막의 두 반쪽 막대와 원뿔 모두에 손상을 입힐 수있는 가장 효과적인 프로토콜입니다. 중요한 것은,이 치료는 색소 동물뿐만 아니라 흰둥이 동물 효과적입니다. 우리는 더 광범위하고 ...

빛에 의한 망막 변성 (LIRD)는 일반적으로 설치류 피해로드와 콘 광수에 제브라 피쉬 모두에 사용됩니다. 성인 제브라 피쉬에서 시세포 변성 뮐러 아교 세포를 다시 입력 세포주기에 트리거 및 과도 증폭 조상을 생산하고 있습니다. 이 조상은 그들이 궁극적으로 새로운 광 수용체를 야기 손상된 영역에 마이그레이션으로 증식을 계속합니다. 현재, 두 널리 사용되는 LIRD 패러다임, 각각의 재생 반응 광수 손실 및 해당 차이의 정도를 변화 결과.가있다 더 많은 유전자와 약물 도구를 재생하는 동안 관심의 개별 유전자의 역할을 테스트 할 수있는만큼, 강력한 LIRD 패러다임을 개발할 필요가있다. 여기에서 우리는 LIRD 프로토콜을 설명하는 우리는 두 가지 이전에 설립 LIRD 기술의 사용을 결합했다하는로드와 콘 광수 모두의 광범위하고 일관된 손실됩니다. 게다가이 프로토콜은 LIRD 연구에 대한 흰둥이 배경에 관심 유전자 변형 라인을 유지하기 위해 필요가 없습니다 색소 동물에 사용하기 위해 확장 할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV light source</td>
			<td style="width:215px;">Leica</td>
			<td style="width:155px;">EL600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Petri dish (150 x 20 mm)</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS3160152BO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 ml glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS1000250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 L glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS10004L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-105</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 W halogen lamps</td>
			<td>Workforce</td>
			<td>265-669</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tanks</td>
			<td>Aquaneering</td>
			<td>ZT180T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tank lids</td>
			<td>Aquaneering</td>
			<td>ZT180LCL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fan</td>
			<td>Honeywell</td>
			<td>HT-900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aerator</td>
			<td>Tetra</td>
			<td>77853-900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermometer</td>
			<td>Cole-Parmer</td>
			<td>YO-08008-58</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bent forceps (5/45)</td>
			<td>World Precision Instruments</td>
			<td>504155</td>
		</tr>
	</tbody>
</table>

a novel light damage paradigm for use in retinal regeneration studies in adult zebrafish

빛에 의한 망막 변성 (LIRD)는 일반적으로 설치류 피해로드와 콘 광수에 제브라 피쉬 모두에 사용됩니다. 성인 제브라 피쉬에서 시세포 변성 뮐러 아교 세포를 다시 입력 세포주기에 트리거 및 과도 증폭 조상을 생산하고 있습니다. 이 조상은 그들이 궁극적으로 새로운 광 수용체를 야기 손상된 영역에 마이그레이션으로 증식을 계속합니다. 현재, 두 널리 사용되는 LIRD 패러다임, 각각의 재생 반응 광수 손실 및 해당 차이의 정도를 변화 결과.가있다 더 많은 유전자와 약물 도구를 재생하는 동안 관심의 개별 유전자의 역할을 테스트 할 수있는만큼, 강력한 LIRD 패러다임을 개발할 필요가있다. 여기에서 우리는 LIRD 프로토콜을 설명하는 우리는 두 가지 이전에 설립 LIRD 기술의 사용을 결합했다하는로드와 콘 광수 모두의 광범위하고 일관된 손실됩니다. 게다가이 프로토콜은 LIRD 연구에 대한 흰둥이 배경에 관심 유전자 변형 라인을 유지하기 위해 필요가 없습니다 색소 동물에 사용하기 위해 확장 할 수 있습니다.

지금까지 설명한 가벼운 치료 프로토콜은 LIRD의 각 방법을 비교 하​​였다. 어두운 처리 성인 흰둥이 동물 (그림 3-5)에서 개별 가벼운 치료는 크게로드의 손실 (그림 3)와 콘 (그림 4) 광수습니다. 그러나 두 개별 치료는 주로 비교적 가벼운 치료 (그림 3과 4)에서 보호 복부 망막을 떠나, 망막 등의 반 광수 손상. 또한, 할로겐 라이트 치료에 ...

Watch this Scientific Journal Video about A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish at JoVE.com

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

복수의 가벼운 손상 프로토콜은 손상 광수를 설명하고 결과적으로 성인 zebrafish에있는 망막 재생 반응을 유도하고 있습니다. 이 프로토콜은 안료 동물에서 사용할 수있는 개선 된 방법을 설명하고 그로드하고 전체 망막에 걸쳐 콘 광수의 대부분을 손상.

성인 Zebrafish의 망막 재생 연구에있는 사용을위한 비발 한 가벼운 손상 패러다임

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

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JoVE Journal

Neuroscience

10.1K 조회수.  Wayne State University School of Medicine.  복수의 가벼운 손상 프로토콜은 손상 광수를 설명하고 결과적으로 성인 zebrafish에있는 망막 재생 반응을 유도하고 있습니다. 이 프로토콜은 안료 동물에서 사용할 수있는 개선 된 방법을 설명하고 그로드하고 전체 망막에 걸쳐 콘 광수의 대부분을 손상. 

비디오: A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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신경과학

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

작은 동물의 만성 신경 레코딩을위한 초경량 동력 Microdrive의 설계 및 조립

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

쥐에 활동 륜 실행에 녹음 및 Circadian 리듬 분석

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

형광 염료와 성인 Zebrafish의 망막 신경절 세포의 퇴행성 라벨링

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

태아 알코올 스펙트럼 장애의 쥐 모델에서 Hippocampal 기능 장애를 평가 하기 위해 추적 Eyeblink 클래식 컨디셔닝의 사용

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

아 밀 로이드-β42 독성 그리고 Zebrafish 두뇌에서 Neurodegeneration 모델링

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

레이저 유도 된 망막 변성 및 재생 Zebrafish 모델 뮐러 명과 셀 활성화

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

Ex Vivo 실험 이미징에 대 한 성인 제 브라에서 준비 신선한 망막 조각

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

두 광자 레이저와 Zebrafish 애벌레의 칼슘 이미징 및 행동 녹화를 사용 하 여 평가 사용 하 여 신경 인구의 제거

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

모델을 새로운 시험관에 폭발 외상 성 뇌 손상의

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...