이 작품은 NIH 교부금 어이 012,031 (동부 표준시 - A), 실명, 주식 회사 (MAZ), 뉴저지 라이온스 안구 연구 재단 (MAZ)을 방지하기 위해 FM 커비 재단 (동부 표준시 - A), 연구에서 수상에 의해 지원되었다 뉴저지 (MAZ), 조셉 J.와 마거리트 DiSepio 망막 연구 기금 (MAZ)와 재니스 미첼 바사르 및 애쉬비 미첼 원정대 (AMK)의 아이 연구소.

1. 세포 배양 삽입에 대한 사용자 지정 만든 스탠드 만들기 5 ML 피펫 팁 (ISC BioExpress, # P - 3250-19)의 기본 완벽하게 10mm의 바닥 크기 (12.6 mm, 외경) (내경) NUNC 세포와 일치하는 13mm의 내경을 문화 삽입 (8 μm의, 폴리 카보 네이트 멤브레인, 열, # 137443). 스탠드를 만들기 위해 피펫은 6mm 롱 홀로 실린더를 생산하는 기지 근처에 차단했다. 그런 다음 조각은 약 5mm로 삽입의 높이를 제기 반지 (높이 2 ㎜), 아래의 세 다리 (높이 4mm)를 생성하기 위해 불꽃 - 가열 칼날을 사용하여 실린더의 측면에서 제거되었습니다. 2. 돼지의 망막의 부착과 문화에 대한 살균 필터 종이 준비 왓먼 4M 필터 용지 (CAT 번호 1004)는 (같은 살균 또는 일단 망막이 첨부되었습니다 유리관에 여과지를 이동에 사용했던 작은 삼각 손잡이 원형 모양 (직경 6.3 ㎜)으로 절단되었습니다 ) 동영상에 표시됩니다. 3. 망막 explants의 준비 5 아이즈 - 150~230파운드 오래된 9 개월은 미국 요크셔 돼지는 핵의 제거 3 시간 이내에 현지 도살장 (얼음 이송)에서 얻은되었습니다. 도착되면, 눈이 관계없는 조직의 청소했다, betadine 솔루션 (10 % Povidone - 요오드, 퍼듀 Frederique 회사, 스탬 포드, CT)에 담근과 Dulbecco의 변형 이글 중간 (1g / L 포도당과 DMEM, L - 글루타민 두 번 씻어 나트륨 pyruvate, Cellgro - mediatech 주식 회사, Manasses, VA)은 2.5 μg / ML Amphotericin B (Gibco - Invitrogen, 칼스 배드, CA)과 보완. 앞쪽에 세그먼트는 신경 망막를 노출, 제거되었습니다. 식스 - mm trephine 블레이드 (Storz 안과 - 바우와 롬, 맨체스터, MO)는 적도, 전체 두께 후부 세그먼트 explants를 잘라하는 데 사용되었습니다. 망막은 이후에 부드럽게, 신경절 세포 레이어에 건조 살균 필터 종이를 적용 신경 망막을 운반하고, 문화 삽입에 부착된 망막과 필터 종이를 배치, photoreceptors가 직면한에 의해 해제 벗겨했다. 삽입은 다음 12 잘 문화 요리 (피셔)에서 완벽하게 망막을 커버해야하고, 문화 매체에 게재되었습니다. 여러 explants는 일상적으로 취득하고 하나의 시선에서 교양되었습니다. 4. 조직 문화 망막 조직은 (멤, Invitrogen - Gibco - 생명 기술 주식 회사, 락빌, MD), 산도 7.4, 0.2 MG / ML 글루타민, 10 μg / ML 돼지의의 인슐린, 1 ㎜의 pyruvate, 0.1와 보충 이글의 최소 필수 매체 교양 MM L - 아스코르비 산, 100 U / ML 페니실린, 100 μg / ML 스트렙토 마이신, 250 NG / ML amphotericin B (Antimycotic 항생제 : 시그마, 세인트 루이스, MO), 그리고 humidified 5% CO 2, 균형 공기와 aerated, 37 ° C. 중간 이틀마다 교환했다. 조직은, 사용 시약 또는 프로브의 다양한 치료, 또는 이후 생화학 또는 형태학의 분석에 대한 치료 남아있을 수 있습니다. 아래의 섹션은 일관성있는 전체 두께 망막 크로스 섹션을 만드는 절차를 설명합니다. 5. Sectioning <ol><li> 그라 이오 스탯을 사용하여 <ol><li> 4 밤새 4 % parafomaldehyde에서 망막 조직을 수정 ° C; </li><li> 인산염은 생리 (PBS)를 버퍼로 간략하게 조직을 씻어; </li><li> 그것은 해부 현미경을 사용하여 35mm 접시에 PBS에있는 동안 조심스럽게 여과지에서 조직을 제거; </li><li> 30 % 자당에 조직을 전송하고 4 박 유지 ° C; </li><li> 멀리 흡입 조직 주변의 과잉 솔루션은 조직을 커버하기에 충분한 조직 테크 매체 (OCT 컴파 운드 4,583) 추가하고 조직을 침투하기 위해 2 시간 동안 실온에서 흡수하게; </li><li> 치과 왁스와 사각 프레임 (10mm X 10mm, 5mm 높이)를 구축하고 조직 주위에 그것을 장소, 냉동실에 프레임 (조직 샘플 평면 있는지 확인)과 자리를 채우기 위해 더 많은 조직 테크 미디어를 추가 (- 20 ° C) 적어도 1 시간; </li><li> 샘플 플랫폼 (샘플 블록은 플랫폼에 수직으로되어 있는지 확인)에 냉동 샘플 블록을 전송; </li><li> 예비 냉각 그라 이오 스탯의 예제 플랫폼을 마운트 (-20 ° C, Leica, CM1900)와 블록이 블레이드에 수직으로되어 있는지 확인하십시오; </li><li> 원하는 두께의 조직을 sectioning 전에 왁스와 프레임 블록을 제거합니다; </li><li> 젤라틴 - 처리 슬라이드에있는 섹션을 놓으십시오. </li></ol></li><li> Vibratome를 사용 <ol><li> 수정 및 그라 이오 스탯을 위해 위에 설명된대로 조직을 씻어; </li><li> 작은 용기에 미리 가열, 녹인 4 % 한천로 조직을 놓고 ° C 한천 확대해까지 완전히 4 유지; </li><li> 내부 조직 샘플과 작은 블록으로 한천을 장식하기 위해 날카로운 칼날을 사용; </li><li> Krazy 접착제와 vibratome 샘플 홀더 (조직 샘플의 조각 세기의 플랫폼에 수직으로되어 있는지 확인에 접착제 샘플 블록E 샘플 홀더, 즉 수직 블레이드)와 완전히 얼음 물 속에 빠져들 샘플 블록과 vibratome sectioning 시스템 (Leica, 시리즈 1000)의 보유자 마운트; </li><li> 50 μm의 섹션에 샘플 블록을 잘라 부드러운 브러시로 젤라틴 - 대우 슬라이드에 올려 놓으십시오. </li></ol></li></ol> 6. Immunohistochemistry 모든 표준 프로토콜을 사용합니다. Immunolabeled 두께의 섹션은 공촛점 현미경으로 볼 수 있습니다. 7. 대표 결과 : 형태는 문화의 7 일의 기간 동안 보존되었다. culturing 전후 망막의 이미지는 동영상에서 사용할 수 있습니다.

<ol>
	<li>Johnson, T. V., Martin, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+an+adult+retinal+explant+organotypic+tissue+culture+system+as+an+in+vitro+intraocular+stem+cell+transplantation+model.">Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model.</a> Invest Ophthalmol Vis Sci. 49, 3503-3512 (2008).</li><li>Kretz, A., Marticke, J. K., Happold, C. J., Schmeer, C., Isenmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primary+culture+technique+of+adult+retina+for+regeneration+studies+on+adult+CNS+neurons.">A primary culture technique of adult retina for regeneration studies on adult CNS neurons.</a> Nat Protoc. 2, 131-140 (2007).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nat Protoc. 1, 2710-2718 (2006).</li><li>Koizumi, A., Zeck, G., Ben, Y., Masland, R. H., Jakobs, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+physiologically+functional+adult+mammalian+retinas.">Organotypic culture of physiologically functional adult mammalian retinas.</a> PLoS One. 2, e221-e221 (2007).</li><li>Lye, M. H., Jakobs, T. C., Masland, R. H., Koizumi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+culture+of+adult+rabbit+retina.">Organotypic culture of adult rabbit retina.</a> J Vis Exp. , (2007).</li><li>Ames, A., Li, Y. Y., Heher, E. C., Kimble, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+rabbit+retina+as+related+to+function:+high+cost+of+Na++transport.">Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.</a> J Neurosci. 12, 840-853 (1992).</li><li>Kobuch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+adult+porcine+retina+and+retinal+pigment+epithelium+in+perfusion+culture:+characterisation+of+an+organotypic+in+vitro+model.">Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: characterisation of an organotypic in vitro model.</a> Exp Eye Res. 86, 661-668 (2008).</li><li>Kaempf, S., Walter, P., Salz, A. K., Thumann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+organotypic+culture+model+of+adult+mammalian+neurosensory+retina+in+co-culture+with+retinal+pigment+epithelium.">Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.</a> J Neurosci Methods. 173, 47-58 (2008).</li><li>Guduric-Fuchs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+pig+retinal+development.">Immunohistochemical study of pig retinal development.</a> Mol Vis. 15, 1915-1928 (2009).</li><li>Chandler, M. J., Smith, P. J., Samuelson, D. A., MacKay, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+density+of+the+domestic+pig+retina.">Photoreceptor density of the domestic pig retina.</a> Vet Ophthalmol. 2, 179-184 (1999).</li></ol>

관심 없음 충돌 선언하지 않습니다.

비전 연구자는 망막 줄기 세포 이식 1, 망막 재생 2, 외인성 유전자 발현 3-5로 문제의 다양한 연구를 organotypic 시스템을 포함한 망막 문화 시스템의 다양한 설립했습니다. 그러나, 성인 포유류의 망막은 체외에서 photoreceptors 6 높은 에너지 요구에 주로 인해 유지하기 어렵습니다. 고이즈미 외가. photoreceptors에 대한 산소 공급이 문화 삽입의 높이를 제기?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 멤 </td><td> Invitrogen - Gibco </td><td> 10370-021 </td><td></td></tr><tr><td> 5 ML 피펫 팁 </td><td> ISC BioExpress </td><td> P - 3250-19 </td><td></td></tr><tr><td> NUNC의 세포 배양 삽입 </td><td> 열 </td><td> 137443 </td><td> 8μm, 폴리 카보 네이트 </td></tr><tr><td> 왓먼의 4M 필터 용지 </td><td> 워트 먼지 </td><td> 1004 </td><td></td></tr><tr><td> DMEM </td><td> Cellgro - mediatech 병원 </td><td> 10-013 - CV </td><td></td></tr><tr><td> 한천 </td><td> Fluka </td><td> 05,040 </td><td></td></tr><tr><td> Fungizone Amphotericin B </td><td> Invitrogen - Gibco </td><td> 15290018 </td><td></td></tr><tr><td> Trephine 블레이드 </td><td> 바우와 롬 </td><td> T3096 </td><td> 6.00mm </td></tr><tr><td> OCT 컴파 운드 </td><td> 전자 현미경 과학 </td><td> 62550-01 </td><td> 4583 </td></tr><tr><td></td><td> 과학 </td><td></td><td></td></tr><tr><td> 그라 이오 스탯 </td><td> Leica </td><td> CM1900 </td><td></td></tr><tr><td> Vibratome </td><td> Leica </td><td> 시리즈 1000 </td><td></td></tr><tr><td> 공촛점 현미경; </td><td> 칼 자이스 혈구 </td><td> LSM510 </td><td></td></tr><tr><td> 안티 - 시냅틱 소포의 단백질 2 (SV2) 마우스 단클론 항체 </td><td> DSHB </td><td> N / A </td><td></td></tr><tr><td> 안티 Glial Fibrillary 산성 단백질 (GFAP) 토끼 polyclonal 항체 </td><td> DAKO </td><td> DAKO </td><td></td></tr><tr><td> Propidium의 이우 (PI) </td><td> 시그마 </td><td> P4170 </td><td></td></tr></tbody></table>

organotypic culture of full-thickness adult porcine retina

동물 실험을 바꾸거나 줄일 수 있습니다 체외 모델에 대한 인식 수요가있다. 돼지의의 망막은 인간의 망막과 유사한 구조의 연결을 가지고 있으며, 따라서 인간의 망막 부상 퇴행성 질병의 메커니즘을 연구에 대한 가치있는 수종입니다. 여기서 우리는 도살장에서 얻은 눈에에서 분리된 돼지의 성인의 망막의 organotypic 문화 비용 효율적인 기술을 설명합니다. 앞쪽에 세그먼트를 제거 후 trephine 블레이드는 성인 돼지의 눈을의 후부 세그먼트에서 여러 신경 망막 - Bruch의 막이 - RPE - 맥락막 - 공막엔의 explants를 만드는 데 사용되었다. 무균 필터 종이 각 explant에서 신경 망막 떠 사용되었습니다. 필터 종이 망막 복합은 맞춤 제작 스탠드로 문화 접시의 바닥에서 거리가 개최되었습니다 삽입, 위에 (최대 photoreceptor 사이드) 교양되었습니다. 스탠드는 망막의 양쪽에 문화 매체를 잘 순환 수 있습니다. 전반적으로,이 절차는 재현성, 간단하고, 적어도 7 일 동안 기본 망막 구조의 보존을 허용, 그것 포유 동물 망막에서 형태학의 약리 및 생화학 연구의 다양한 유용 모델 만들기.

Watch this Scientific Journal Video about Organotypic Culture of Full-thickness Adult Porcine Retina at JoVE.com

Organotypic Culture of Full-thickness Adult Porcine Retina

여기 우리는 일주일 동안 성인 돼지의의 망막의 organotypic 문화 비용 효율적인 기술을 설명합니다. 간단히, 멸균 여과지는 맞춤 제작 스탠드 제기 삽입에 RPE 및 장소 photoreceptor 사이드 최대의 신경 망막 떠 사용되었습니다.

전체 두께 성인 돼지의의 망막의 Organotypic 문화

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

organotypic-culture-of-full-thickness-adult-porcine-retina

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JoVE Journal

Neuroscience

14.9K 조회수.  University of Medicine and Dentistry of New Jersey - UMDNJ. 여기 우리는 일주일 동안 성인 돼지의의 망막의 organotypic 문화 비용 효율적인 기술을 설명합니다. 간단히, 멸균 여과지는 맞춤 제작 스탠드 제기 삽입에 RPE 및 장소 photoreceptor 사이드 최대의 신경 망막 떠 사용되었습니다.

비디오: Organotypic Culture of Full-thickness Adult Porcine Retina

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

의 연결을 부상을 검사에 대한 Organotypic Hippocampal 슬라이스 문화 모델

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

연구

신경과학

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

의 Organotypic 슬라이스 문화 GFP - 표현 주변 신경 가지의 실시간 이미징을위한 마우스 배아를

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

개발 마우스 망막의 생체내 Electroporation에

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

전기 생리 연구 성인 마우스 망막의 수평 슬라이스의 준비

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

배아 마우스 두뇌의 organotypic 슬라이스 문화에서 마이 그 레이션 뉴런의 저속 공 촛점 이미징<em&gt; Utero에서</em&gt; 일렉트로 포 레이션

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

성인 인간 피부 섬유 아 세포에서 유도 된 신경의 고수익 문화의 간단한 세대

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

성인 쥐 요로 방광에서 1 차신경및 glia의 격리 그리고 문화

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

성인 프레리 볼레스의 신경질성 틈새에서 파생된 신경구 의 문화

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

마우스 해마 흥분및 직업 별 억제 신경의 다른 organotypic 슬라이스 문화에 걸쳐 단세포 전기화

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...