페드로 드 라 바리 마자 재단은이 작품을지지했습니다.

Rotenone 1) Intragastric 관리 <ol><li> 동물의 무게를. </li><li> 필요한 rotenone / 자동차 솔루션의 전체 볼륨을 계산합니다. 동물 중량 g 당 이러한 경우에는 0.01 ML. </li><li> 이전에 계산 rotenone 솔루션의 볼륨 (0.625 MG / ML rotenone, 4 % carboxymethylcellulose 및 1.25 %의 클로로포름) 또는 차량 용액 (4 % carboxymethylcellulose 및 1.25 %의 클로로포름)와 ​​1 ML의 주사기를 부과합니다. 이 경우는 5 MG / kg rotenone 선량에 해당합니다. </li><li> 마우스를 들고, 한 손으로 마우스의 꼬리를 들고, 다른 한편의 첫 번째와 두 번째 손가락으로 당겨 목 피부를 스트레칭. </li><li> 머리가 두 손가락 사이에 고정되면, 손바닥에 대해 그것을 엎드려서 거꾸로 그것을 켜십시오. 네 번째와 다섯째 손가락을 사용하여 꼬리를 수정. </li><li> 뒤로 향해 머리를 유지, 마우스의 입을 열 미각을 누르십시오. 부드럽게 입안에 gavage을 소개합니다. gavage 바늘이 아닌 일반 바늘을 사용하는 것이 중요합니다. 천천히, 멀리 위 방향으로 gavage 이동합니다. 이것은 폐에 받고 자료를 막을 수있을 겁니다. </li><li> 부드럽게 색전증을 눌러 rotenone 솔루션이나 차량을 관리할 수 있습니다. 완료되면, 천천히 gavage 압축을 풉니다. </li><li> 한 케이지에서 모든 동물이 완료되기 전까지 다른 새장에있는 동물을 돌려 놓습니다. </li></ol> 2) Immunostaining 절차 <ol><li> 치료 후, 마우스는 Ketamin 100 MG / kg 놀이 Xylazin 10 MG / kg IP 및 intracardially PBS의 4 % Paraformaldehyde로 perfused를 사용하여 anesthetized 있습니다. </li><li> 배짱은 다음 절개로 추출하고, 야간 15 % 자당에 배치됩니다. 그 다음날은, 조직은 30 % 자당에 전송하고 4 박 다시 incubated 있습니다 ° C. </li><li> 조직은 다음 조직 테크에 위치하고 사용할 때까지 -80 ° C의 냉동고에 저장됩니다. </li><li> 그라 이오 스탯을 사용하여 20 마이크론 소장 교차 섹션은 Superfrost 슬라이드로 전송됩니다. </li><li> 안티 βIII - tubulin 및 안티 알파 synuclein 항체를 사용하여 프로 시저를 차단하고 immunostaining 것은 다음 앞에서 설명한 9로 수행됩니다. </li></ol> 3) 알파 synuclein의 포함 이미지 분석 그것은 이미지가 &#39;원산지 모르고 외부 사람이 분석을 수행하는 것이 좋습니다. 따라서, 데이터는 codified해야합니다. 이미지 분석 피지 소프트웨어를 사용하여 수행되었습니다. <ol><li> 공촛점 이미지 파일을 엽니다. 그것이 개별적으로 각 채널에서 작동이 가능합니다 그래서 그 다음, 채널을 분할. </li><li> 사용하고 Analyse&gt; 규모를 설정하고 1 픽셀 크기를 설정을 선택하지 않을 이들 채널을 닫습니다. 0.13 (이것은 객관적이고 사용되는 해상도에 따라 달라집니다)로 알려진 거리를 설정하고 세계 클릭하십시오. 이미지의 크기는 현재 미크론으로 표시됩니다. </li><li> 알파 synuclein 채널을 선택하고 신경절을 선택, 선택이 모든 조각에 신경을 encloses 있는지 확인하십시오. </li><li> 보도 자료 이미지&gt; 이미지를 복제하는 중복. 신경절을 선택한 단지 이미지는 신경절의 크기가 중복됩니다. </li><li> 보도 편집&gt; 지우기 밖에. 선택 영역 밖으로 이미지의 부분은 검정색이 될 것입니다. </li><li> 프로 시저 간 얼마나 잘 나중 단계에서 확인하기 위해 이미지를 다시 복사. 배경 노이즈 비율에 높은 신호를 가지고 이미지는 이미지 분석을 위해 사용하지 않아야합니다. </li><li> 이미지&gt; 조정&gt; 임계값을 선택하고 다음 MaxEntropy 선택합니다. 그런 다음 프로 시저가 잘 된 것을 보장하기 위해 자른 이미지와 원래의 하나의 슬라이스를 통해 이동합니다. entropic 임계값은 이미지의 히스토그램이 임계값 이상의 농도가 검정색 이미지 위에 흰색에 표시됩니다에서만 해당 픽셀에 따라 강도 신호 레벨을 선택합니다. </li><li> 프로세스를 선택한 다음, 부드러운 클릭하십시오. 이 단계는 매끄러운 가장자리에있는 이미지의 pixeled 가장자리를 변환합니다. </li><li> 프로세스를 선택한 다음 바이너리를 클릭하고, 바이너리를 만들기를 선택합니다. 이미지가 이미지 위에 흰색 검정색으로 표시됩니다. </li><li> 중복 이미지 다음 단계를 수행하는 이미지를 중복. </li><li> 분석&gt; 분석 입자를 클릭하십시오. 그런 다음 표시를 선택 : 개요하고 요약을 클릭합니다. 설정의 나머지 부분은 기본에 잎이 달린해야합니다. 이 함수는 신호와 함께 그룹화하는 기능과 픽셀의 총 개수를 인식합니다. 신호 플러스 픽셀 그룹의 수와 평균 크기가 픽셀의 전체 표면은보고됩니다. </li><li> 잘 조정 가장 큰 조각을 비교하고 선택합니다. 슬라이스가 표시됩니다. </li><li> 총 알파 synuclein 표면, 흠도의 수와 평균 입자 크기의 데이터 테이블을 복사합니다. Excel 스프레드 시트 또는 다른 주석에 데이터를 붙여 넣습니다. </li><li> 피지 (으)로 돌아가기와 β - tubulin 채널에서 이전에 선택한 슬라이스를 찾아, 그것을 선택합니다. </li><li> 신경절 영역을 선택하고을 클릭 다음 측정을 선택 분석. 또 다른 테이블을 보여주는가 나타납니다신경절 총 표면. </li><li> 알파 synuclein 측정 근처 엑셀 시트로 테이블에서 데이터를 추출하거나 다른 곳에서 내려 복사합니다. </li><li> 엑셀 사용 신경절의 크기로 정규화 총 알파 synuclein의 표면 및 포함 번호를 얻으려면 신경절 표면으로 알파 synuclein 측정에서 얻은 여러 가지 매개 변수를 나눕니다. </li></ol> 4) 대표 결과 gavage 관리 프로토콜이 올바르게 수행하면 동물에 대한 불편을 최소화하고 있습니다. rotenone의 농도를 사용하는 치료는 혈액에 rotenone 수준없이 팔에 rotenone의 로컬 효과를 가능하게하고 근육이나 뇌 mitochondrial 콤플렉스없이 억제 나 역시 치료의 1.5 개월 (그림 1 참조) 후. 이미지 분석이 올바르게 수행하는 경우 알파 synuclein 패턴의 엄격한 분석이 가능하여야한다. 그것은 알파 - synuclein (총 표면), 알파 - synuclein의 셀에 패턴 (흠도의 수)와 알파 synuclein의 흠도의 존재 (포함 크기) (그림 2)의 총액에 대한 신뢰할 수있는 데이터를 제공합니다. <img src="/files/ftp_upload/2123/2123fig1.jpg" alt="그림 1" /> 그림 1. rotenone의 모터 장애는 혈액이나 CNS에 rotenone의 검출없이 쥐를 치료. 표준 (50 NG / ML) 20 10 5 MG / kg 취급 생쥐의 두뇌 샘플에서 크로마토 그램. B와 C, rotenone 혈액의 수준 (B) 및 CNS (C)의 양을 정함. B, 혈액 레벨 1 , 치료 후 2, 3 시간. 마우스가 세 그룹 (N = 3)에 나누어 2.5, 5, 10 및 20 MG / kg rotenone 취급했다 (N = 3), 혈액 300 μL는 rotenone 관리 후 1, 2, 3 시간 추출하여 함께 풀링되었습니다 5 (N = 3), 10 (N = 3) 및 20과 함께 하루 (N = 1) MG / kg rotenone, 두뇌 및 brainstem가 2 시간 후에 1을 추출하고 있었다 한번 HPLC 분석. C는 생쥐 한 주 동안 치료를했다 마지막으로 관리 및 HPLC 분석. D, mitochondrial 콤플렉스 I 1.5 개월 치료를 생쥐의 근육과 두뇌 샘플의 활동 준비에 들어갔다. <img src="/files/ftp_upload/2123/2123fig2.jpg" alt="그림 2" /> 그림 2. 로컬로 관리되는 rotenone은 팔의 신경에 gliosis와 알파 synuclein의 인산화, 축적 및 집계를 유도. (스케일 바 20 μm의). A, B, C, 안티 βIII - tubulin, 알파 synuclein과 십이지장에서 DAPI의 얼룩 (B)과 회장 (이 A, C) 부분. 3 개월 제어 (A)에 비해 B의 화살표는, 1.5 개월 치료 장용 신경계의 신경 내부 증가 알파 synuclein 작은 반점이있는 패턴을 유도. C에서 화살표, 큰 알파 synuclein의 함유 (ø&gt; 6 μm의)의 3 개월 치료를 유발 형성. D, 안티 - 알파 - synuclein, Thioflavine S 및 DAPI를 사용하여 immunofluorescence 염색법. D에 화살표, 오직 삼개월 취급 쥐가이 큰 알파 synuclein의 축적의 집합을 보여주었다. E, E &#39;, E&#39;, 이미지 분석 부량 단계. E, 팔의 신경절을 보여주 부량에 사용되는 이미지의 예를 보여주는 알파 synuclein에 대한 immunostained 다른 흠도. E &#39;, 이미지 분석 프로토콜 후 얻은 이미지의 예. E&#39;, 원래 흠도 (녹색)와 프로토콜 (노란색 선)을 수행한 후 얻은 표현의 일부 사이에 중복됩니다. FG에 표시된 실험 부량 AC는 자동으로 세분화 및 엔트로피 기반 thresholding 방법을 사용하여 만들어졌다. 단일 별표, P &lt;0.05, 두 번 별표, P &lt;0.01. F, 각 열의 총 알파 synuclein의 표면 / 신경절 표면을 나타냅니다. G, 각 컬럼은 알파 synuclein의 함유 / 신경절 표면의 총 수를 나타냅니다. 모든 그래프는 ± SEM을 의미 표시

<ol>
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A., Bohl, J., Del Tredici, K., K, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+alpha-synuclein+immunoreactive+inclusions+in+Meissner's+and+Auerbach's+plexuses+in+cases+staged+for+Parkinson's+disease-related+brain+pathology.">Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.</a> Neurosci Lett. 396, 67-72 (2006).</li><li>Wakabayashi, K., Takahashi, H., Ohama, E., Ikuta, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+an+immunohistochemical+study+of+Lewy+body-containing+neurons+in+the+enteric+nervous+system.">Parkinson's disease: an immunohistochemical study of Lewy body-containing neurons in the enteric nervous system.</a> Acta Neuropathol. 79, 581-583 (1990).</li><li>Wakabayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+occurrence+of+Lewy+bodies+in+the+dorsal+vagal+nucleus+in+a+patient+with+late-onset+parkinsonism.">Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.</a> J Neurol Sci. 165, 188-191 (1999).</li><li>Braak, H., Sastre, M., Bohl, J. 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프란 시스코 판 - Montojo이 동물 모델 (신청 번호 PCT / EP 2009/005688)에 대해 보류중인 특허 출원 있습니다.

Intragastric 관리는 이전에 12을 수행하고 있습니다. 그러나, 0.5 % carboxymethylcellulose 나트륨 소금 (CMSS) 혼자 인덴의 원고에 따르면, rotenone가 실시되었다 해산. Rotenone는 높은 liphophylic 물질입니다. 따라서, rotenone 혼자 0.5 % CMSS에 녹아 수 없습니다 그래서 할 경우에는 침전됩니다. 클로로포름의 사용은 강수량을 피한 균등하게 분산 rotenone 서스펜션을 만듭니다. 또한, CMSS에서 살충제의 높은 농도...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>R8875</td>
<td>Germany</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3313.1</td>
<td>Germany</td>
</tr>
<tr>
<td>Carboxymethylcellulose</td>
<td>&nbsp;</td>
<td>SIGMA-Aldrich</td>
<td>C5678</td>
<td>Germany</td>
</tr>
<tr>
<td>Gavage 1,2 mm x 60 mm</td>
<td>&nbsp;</td>
<td>Unimed</td>
<td>&nbsp;</td>
<td>Switzerland</td>
</tr>
<tr>
<td>LSM 510</td>
<td>&nbsp;</td>
<td>Zeiss Microscopes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FIJI</td>
<td>&nbsp;</td>
<td>http://pacific.mpi-cbg.de</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

oral administration of rotenone using a gavage and image analysis of alpha-synuclein inclusions in the enteric nervous system

파킨슨병 (PD) 환자에서 연관된 병리 인터 알리아 장용 신경계 (팔) 1,2, 후각 망울 (OB), 미주 (DMV) 3 등의 모터 핵, intermediolateral 관련된 특성 패턴을 따릅니다 척수 4와 substantia 니그라의 핵, 4,5 질병의 neuropathological 준비를위한 기초를 제공합니다. 팔과 OB가 가장 노출된 신경 구조와 영향을 가장 먼저 사람입니다. 흥미롭게도, PD는 농약 노출 6-8 관련된되었습니다. 여기 세부에서 우리의 이전 연구 9에 사용되는 두 가지 방법을 보여줍니다. 팔에서 로컬로 연기 rotenone의 효과를 분석하기 위해, 우리는 한 세 C57/BL6 마우스로 gavage를 사용하여 rotenone을 관리. Rotenone 강력 mitochondrial 복잡한에게 I 10을 억제 널리 사용되는 살충제이다. 그것은 매우 lipophylic과 저조한 위장관 11 흡수됩니다. 우리의 결과는 5 MG / rotenone의 kg의 행정부가 mitochondrial 복잡한 I 근육이나 두뇌의 활동을 억제하지 않았 것으로 나타났다. 따라서, 우리의 관리 방법 rotenone을 사용하면 hepatoportal 시스템을 교차하지 않았고 오직 팔에 행동 것을 제안. 여기서 우리는 엔터 프라이즈 네트워크에 알파 synuclein의 축적에 살충제의 효과를 분석하는 데 사용하는 gavage 및 이미지 분석 프로토콜을 사용하여 살충제를 관리하는 방법을 보여줍니다. 첫 번째 부분은 원하는 정확한 농도의 살충제 (rotenone)의 intragastric 관리를 허용하는 방법을 보여줍니다. 두 번째 방법은 이미지 분석 소프트웨어를 사용하여 엔터 프라이즈 네트워크에 알파 synuclein의 축적을 분석 반자동 이미지 분석 프로토콜을 보여줍니다.

Watch this Scientific Journal Video about Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System at JoVE.com

Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

파킨슨병은 살충제에 대한 노출과 관련된되었습니다. 여기서 우리는 원하는 농도와 장용 신경계의 알파 synuclein의 축적에서 효과를 분석하는 방법에 위장 튜브를 사용하는 살충제를 전달하는 방법을 보여줍니다.

장용 신경계의 알파 synuclein의 흠도의 Gavage 및 이미지 분석을 사용하여 Rotenone의 구강 관리

In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2, ...

oral-administration-rotenone-using-gavage-image-analysis-alpha

Research

JoVE Journal

Neuroscience

16.8K 조회수.  Technische Universität Dresden. 파킨슨병은 살충제에 대한 노출과 관련된되었습니다. 여기서 우리는 원하는 농도와 장용 신경계의 알파 synuclein의 축적에서 효과를 분석하는 방법에 위장 튜브를 사용하는 살충제를 전달하는 방법을 보여줍니다.

비디오: Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작<em&gt; utero에서</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

연구

신경과학

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

설치류 중추 신경계의 생체 Optogenetic 자극

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

장용 신경계의 제자리 칼슘 이미징에서

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, &#945;-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated &#945;-syn is a neuropathological feature of Parkinson&#39;s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble &#945;-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of &#945;-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated &#945;-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal &#945;-syn biology in transgenic animal models harbouring different &#945;-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.

Here, we present a protocol for the isolation of increasingly insoluble/aggregated alpha-synuclein (&#945;-syn) from post-mortem human brain tissue. Through the utilization of buffers with increasing detergent strength and high-speed ultracentrifugation techniques, the variable properties of &#945;-syn aggregation in diseased and non-diseased tissue can be examined.

파킨슨 뇌에서, 수용성 알파 시누 클레인의 순차 추출

Alpha-synuclein (&#945;-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

알파 - 시누 클레인 브릴의 주변 접종 후 형질 전환 마우스의 신경 염증의 생물 발광 영상

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

메서드 및 Adeno 관련 바이러스를 쥐의 정 맥 관리 및 중앙 신경 전달의 평가 대 한 팁

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

소 대형의 강력한 셀 모델로 기본 신경 하 Microsomes 관련 알파 synuclein 집계의 외 인 관리

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

화학유전학적으로 조작된 피질 인터뉴런 선조를 출생 후 마우스 뇌로 이식

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson&#39;s disease synucleinopathy and nigrostriatal degeneration. The standardization of &#945;-syn PFF generation and in vivo application is critical in order to ensure consistent, robust &#945;-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric &#945;-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of &#945;-syn PFFs into rats or mice. Starting with monomeric &#945;-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed &#945;-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated &#945;-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.&#160;

The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

모노 머 로부터 미리 형성 된 섬유 소의 알파-시 뉴 클레 인 생성 및 생체 내 사용

Use of the in vivo alpha-synuclein preformed fibril (&#945;-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model ...