Name: assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method
Uploaded: 2017-06-22T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

이 연구는 NIH P20 GM109005 (ARA) 및 Translational Neuroscience IDeA 프로그램 상 P30 GM110702 센터의 파일럿 보조금에 의해 지원되었습니다.

실험은 UAMS 기관 동물 관리 및 사용위원회 (Institutional Animal Care and Use Committee)가 승인 한 윤리적 기준에 따라 수행되었습니다. 1. 동물과 5-Fu 주사 패러다임 <ol><li> 6 개월 된 수컷 C57Bl6 / J 야생형 마우스를 구입하여 1 살이 될 때까지 일정한 12 시간의 명암주기하에 보관하십시오. </li><li> 0.9 % 살균 식염수로 5 푸를 희석하십시오. 마우스 당 필요한 용량으로 60 mg / kg을 사용하십?...

<ol>
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Vibroslice NVSL &amp; Vibroslice NVSLM123 Available from: <a target="_blank" href="https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf">https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf</a> (2000)</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurolucida 11.03. , (2017).</li><li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+in+the+neurons+of+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization in the neurons of the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Pillai, A. 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보다 현대적인 기술에 비해 Golgi-Cox 방법은 척추 형태를 검사하는 데 선호되는 몇 가지 장점이 있습니다. 1) 염색은 본질적으로 모든 조직에 사용할 수 있습니다. 2) 기본적인 광학 현미경 설정만으로 모든 것이 가능합니다. 3) Golgi-Cox 이미징은 공 촛점 영상보다 빠르다. 4) Golgi 염색 된 절편은 형광 라벨이 부착 된 샘플보다 몇 개월에서 몇 년 더 오래 실행 가능하다. 이러한 장점을 가지고 있어도 Gol...

<html> Dendrites는 presynaptic 입력을 받아 처리하는 뉴런의 가장 큰 부분입니다 1 . 그들의 수지상 프로세스는 복잡한 기하학을 가지고 있는데, 근위부 가지가 원위부 가지보다 더 큰 직경을 가지고 있습니다. 수상 돌기가 발달함에 따라 돌기 arborization이라고하는 과정에서 다른 뉴런과 여러 연결을 형성합니다. 이 분기의 범위와 패턴은 수상 돌기가 적절하게 처리 할 수있는 시냅스 입력의 양을 결정합니다 2 . 수상 돌기 arborization은 활동 의존성 소성과 연결 회로의 적절한 개발을 위해 필요한 과정입니다. 확장, 후퇴, 분지 및 시냅스 생성은 내인성 유전 프로그램 및 외인성 요인으로부터의 영향을 포함하는 복잡한 과정이다. 해마 내의 신경성 수상 돌기의 형태 및 분지 변화는인지 및 기억 형성에 관련되어있다돌기 복잡성의 변화는 병리 생리학 및 행동 변화와 관련이있다 5. 이상은 X- 증후군 및 다운 증후군을 포함한 여러 질병 상태와 관련이있다. 수상 돌기 (dendritic spines)는 중추 신경계 내에서 흥분성 입력을받는 돌기 아버의 특화된 세포 내 구획입니다. 각 계통의 크기와 모양에 따라 3 가지 형태의 계통 분류가 있습니다. 1) 버섯 계통은 다른 계통보다 글루타메이트 수용체가 많은 복잡한 계피 밀도를 가지고 있습니다. 2) 줄기가없는 멍한 등뼈; 3) 길고 좁은 줄기와 구상 머리로 구성된 얇은 등뼈 8 . 돌기 척추 부피는 부분적으로 사용되어 얇은 척추가 일반적으로 더 작습니다 (0.01 μm 3 </sup&gt;)를 버섯 등뼈 (0.8 μm 3 )와 비교 9,10 . 등뼈는 성숙과 함께 안정됩니다. 예를 들어, 얇은 등뼈는 며칠 후에 수축되거나 버섯 등뼈로 발전합니다. 양자 택일로, 버섯 등뼈는 상대적으로 안정하고 오랜 기간 생존 할 수 있습니다. 연결 연결의 강도는 등뼈 및 / 또는 그들의 볼륨 11,12,13의 수를 기반으로 생각됩니다. 고전적인 Golgi 염색 방법과 그보다 현대적인 변형 모두 돌기의 척추 형태와 밀도를 검사하는데 유용합니다. Golgi 염색의 한 가지 독특한 측면은 무작위로 총 뉴런의 약 5 %를 얼룩이 지므로 개별 뉴런의 추적이 가능합니다 14 , 15 . 골지기의 정확한 메카니즘얼룩 개별 뉴런은 여전히 ​​알려져 있지 않습니다, 방법의 원리는은 크로메이트 (Ag 2 CrO 4 ) 16 , 17 의 결정화를 기반으로합니다. Golgi 방법에는 세 가지 주요 유형이 있습니다 : 빠른 골지, Golgi-Cox 및 Golgi-Kopsch 18 , 19 . 세 가지 방법 모두 몇 일에서 몇 달 동안 크롬 염에서 초기 인큐베이션 단계로 시작하지만 몇 가지 주요 차이점이 있습니다. 빠른 Golgi는 첫 번째 단계에서 오스뮴 tetroxide를 사용하는 반면, Golgi-Kopsch는 paraformaldehyde를 포함합니다. rapid-Golgi와 Golgi-Kopsch에서의 염색은 약 7 일 동안 1 ~ 2 % 질산은 용액에서 배양한다. Golgi-Cox 방법은 질산은 대신에 염화 수은과 중크롬산 칼륨을 사용하며 2 ~ 4 주간의 함침 시간이 있습니다. 그 다음 조직을 절편 화하고 희석 된 암모니아염료를 제거하기위한 사진 용 정착액이 뒤 따른다. 세 가지 유형 중에서 골지 - 콕스 (Golgi-Cox) 방법은 배경 간섭이없는 돌기 아버를 염색하는 데 가장 좋은 것으로 여겨지는데 부분적으로는 크리스탈 아티팩트가 조직 표면에 발생하지 않기 때문에 (빠른 골지 방법과 달리) 17 , 20 , 21 . 현재의 방법은 상업적 골지 염색 키트와 함께 제공되는 프로토콜의 약간의 변형이며 5-Fu의 비교적 적은 양이 수지상 형태 학적 특성 및 척추 밀도에 어떻게 영향을 미치는지 평가하기 위해 고안되었다. 획득 된 모든 데이터는 화학 요법 치료가 어떻게 신경 회로에 영향을 미치는지에 대한 더 많은 통찰력을 제공 할 수 있습니다.

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			<td height="21" style="height:21px;width:216px;">superGolgi Kit&#160;</td>
			<td style="width:207px;">Bioenno Lifesciences</td>
			<td style="width:119px;">30100</td>
			<td style="width:216px;">&#160;Contains hazardous materials.&#160;</td>
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			<td height="21" style="height:21px;width:216px;">PBS 10X powder concentrate</td>
			<td style="width:207px;">Fisher&#160;</td>
			<td style="width:119px;">BP665-1</td>
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			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">9002-93-1</td>
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			<td height="21" style="height:21px;width:216px;">Permount&#160;</td>
			<td style="width:207px;">Fisher&#160;</td>
			<td style="width:119px;">SP 15-100</td>
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			<td height="21" style="height:21px;width:216px;">Slide cover&#160;</td>
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			<td style="width:119px;">12-546-14</td>
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			<td height="21" style="height:21px;width:216px;">7mL Transfer pipette&#160;</td>
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			<td style="width:119px;">135030</td>
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			<td height="21" style="height:21px;width:216px;">10 mL Falcon tubes&#160;</td>
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			<td style="width:119px;">352099</td>
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			<td height="21" style="height:21px;width:216px;">Foil&#160;</td>
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			<td style="width:119px;">01-213-105</td>
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assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method

수상 돌기는 흥분성 시냅스를 포함하는 신경 돌기 샤프트의 돌기입니다. 해마 내의 신경성 수상 돌기의 형태 및 분지 변이는인지 및 기억 형성에 관여한다. Golgi 염색에 대한 몇 가지 접근법이 있으며, 모두 수지상 세포의 형태 학적 특성을 결정하고 명확한 배경을 생성하는데 유용합니다. 현재 Golgi-Cox 방법 (상업적 골지 염색 키트와 함께 제공되는 프로토콜의 약간의 변형)은 비교적 낮은 용량의 화학 요법 약물 인 5- 플루오로 우라실 (5-Fu)이 수지상 형태에 어떻게 영향을 미치는지 평가하기 위해 고안되었다 , 척추의 수 및 해마 안의 정강이 형성의 복잡성과 관련이 있습니다. 5-Fu는 수지상 복합체를 상당히 변조 시켰고, 해마 전역에서 척추 밀도를 영역 특이 적 방식으로 감소시켰다. 제시된 데이터는 Golgi 염색 방법이CA1, CA3 및 해마의 치아 이랑 (dentate gyrus)에서 성숙한 뉴런을 fectively 염색했다. 이 프로토콜은 각 단계의 세부 사항을보고하여 다른 연구자가 뇌 전체에 걸쳐 조직을 안정적으로 얼룩지게하고 고품질의 결과와 최소한의 문제 해결을 가능하게합니다.

수상 돌기 arborization에 5 -Fu 처리의 효력 및 Golgi 얼룩 두뇌 단면의 해마에있는 복합성은 상업적으로 이용 가능한 화상 진찰 소프트웨어를 사용하여 정량되고 추적되었다. 추적 후, 돌기 arborization, 척추 밀도 및 척추 형태학은 Sholl 분석 및 수지상 복합 지수 (DCI)를 사용하여 분석되었습니다. Sholl 분석은 수지상 세포 형태를 결정하는 데 사용할 수있는 정량 분석 ​​방법입?...

Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

여기서 우리는 Golgi-Cox 프로토콜에 대해 자세히 설명합니다. 이 신뢰할 수있는 조직 얼룩 방법은 최소한의 문제 해결과 함께 해마 및 전체 뇌에 걸쳐 cytoarchitecture의 높은 품질 평가를 허용합니다.

골지 - 콕스 방법을 이용한 고령의 해마 상피 세포의 수지상 돌기 복잡성 평가

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

The overall goal of this methodology is to properly Golgi stain brain tissue in order to examine and quantify dendritic morphology and complexity. This method can help answer key questions in neuroscience. Since Golgi staining only stains a small subset of cells, this technique could be used to trace the structure of neurons.The main advantage of this technique is that brain sections can be easily stained without risk to them drying out or falling off during any of the washing steps. Demonstrating the procedure will be Fred Kiffer, Tyler Alexander and Julie Anderson, graduate students in Dr.Allen's laboratory. Begin by immersing freshly harvested, unfixed rodent half brains in five milliliters or mercuric chloride-based solution.As mercuric chloride solution is light sensitive, cover the tube with foil. Store the samples in the dark at room temperature. The following day, slowly decant the solution into a temporary container before properly disposing of it in a biohazard waste container.Add five milliliters of fresh mercuric chloride solution and ensure that the brains are immersed. Return the samples to the dark at room temperature for 13 more days. After 14 days, prepare post-impregnation buffer by adding 30 grams of powered post-impregnation buffer to 90 milliliters of distilled or deionized water, DH2O.Fill each well of the six-well plate with 6.5 milliliters of the post-impregnation buffer, one well per brain. Then, remove the mercuric chloride solution from the brain tissue as before. Rinse the tissue with DH2O, then transfer the tissue to the six-well plates with post-impregnation buffer.Cover the plates with foil and store at room temperature in the dark. The next day, aspirate and replace the post-impregnation buffer. Re-cover the plate and store in a four degree Celsius fridge.Prepare two or three 12-well plates per brain by first labeling the plates on their lids with numbers in ascending order from left to right and up to down. Add the identification number of the brain sectioned. Pipette two milliliters of 1X PBS into each well of each plate, then set up the stage and turn on the vibratome.Then, put tape on the tissue block of the specimen holder and add a little cyanoacrylate glue on top. Remove a brain from the six-well plate and place it on a plastic or glass dish. Use a stainless steel double edged blade to cut the cerebellum caudally, making sure that the portion of the cerebellum remaining on the brain is flat.Then, place the flat surface of the remaining cerebellum on the glue. After the glue is dry, place the specimen holder with the tissue into the specimen bath. Place the specimen bath on the vibratome and cover the tissue sample in PBS.Then, attach the blade to the blade holder of the vibratome and slowly lower the blade into the specimen bath until it is fully submerged and slightly below the top of the tissue. Next, set the vibratome speed to seven, the amplitude to six and the cutting angle to 12 degrees. Start the vibration and cut 200-micron sections.Use a large paint brush to move the tissue sections from the specimen bath into each designated well of the 12-well plates. Continue to cut the tissue until the desired number of tissue sections is reached. After sectioning, use a transfer pipette to aspirate the PBS from the wells of the 12-well plate, then add two milliliters of PBS-T and wash for 30 minutes with agitation.Close to the end of the wash period, dilute three parts ammonium hydroxide solution with five parts DH2O. Then, after removing the PBS-T, add the diluted ammonium hydroxide solution. Protect from light using foil and stain the sections for 19 to 21 minutes.Then, after safely removing the ammonium hydroxide solution, add post-staining buffer. Protect with foil and stain for another 19 to 21 minutes. Finally, rinse the sections in PBS-T three times for five to 10 minutes each time.Mount the sections on 1%gelatin coated slides with the large paint brush. Allow them to dry for 20 to 30 minutes at room temperature, then place them in a Coplin Jar and leave overnight. Remove the slides from the Coplin Jar and place them in a plastic rack in the fume hood.Place in a staining dish containing 100%ethanol for five minutes. Dehydrate the sections through two further five-minute ethanol washes. Next, place the slides in a glass rack and use a metal handle to lower the rack into a glass staining dish containing 99%xylene.Wash the slides in two changes of xylene for five minutes each. After the time for the second wash has elapsed, remove the slides from the xylene one at a time and quickly cover the tissue with approximately 0.25 milliliters of mounting media. Next, take a slide cover and lay it over the media.Push any trapped air bubbles underneath the slide covers to the edge with a blunt object, such as the end of paint brush. Place the slides in an area out of direct sunlight and allow them to dry for two to three days. After drying, proceed to image acquisition using a microscope and analysis with the appropriate software.This bright field image shows a Golgi-stained neuron that meets the criteria for imaging and further analysis. All three spine types can easily be seen. Staining is consistent across the entire length of the dendrite and the dendrite is isolated from other neurons.This figure shows that Golgi stained neurons within the Dentate Gyrus showed decreased length at the fourth and sixth order following 5-fluorouracil treatment compared to the saline-treated group. Golgi staining can be used to detect significant differences in spine density. In the CA1 apical pyramidal dendrites, 5-fluorouracil administration to live mice significantly decreased spine density.In the basal dendrites, there were no significant changes in the overall density of spines. Sholl Analysis of CA1 apical pyramidal dendrites revealed that 5-fluorouracil treatment significantly decreased dendritic arborization at distances between 40 to 160 microns away from the soma. In the basal dendrites, the 5-fluorouracil treatment decreased the dendritic arborization at 30 to 110 microns away from the soma.Once mastered, this technique, accounting for both the Golgi staining process and mounting, cleaning and covering can be done in three hours per brain if performed properly. After its development, this technique paved the way for researchers in the field of neurobiology to explore neuroanatomical connections in a murine model. While attempting this procedure, it's important to remember to keep the sections in ammonium hydroxide solution for only 19 to 21 minutes as to not overstain the neurons.After watching this video, you should have a good understanding of how to properly stain, section and rinse brain tissue. Don't forget that working with the mercuric chloride-based solution, ammonium hydroxide solution and xylene can be extremely hazardous, so take precautions and wear lab gloves and work in a fume hood when performing this procedure.

Research

JoVE Journal

Neuroscience

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마우스에 모터 밸런스 조정의 평가는 밸런스 빔을 사용하여

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연구

신경과학

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

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FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

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Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

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Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

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