이 작품은 신경 장애 및 뇌졸중 NIH 국립 연구소 (NINDS) (NS049442)에 의해 지원되었다.&gt;

Resuspending β - 아밀로이드 펩타이드 시작하기 전에 준비했습니다 : <ul><li> Aβ 1-42 (동결 건조된 분말) </li><li> 1,1,1,3,3,3 - Hexafluoro - 2 - 프로파놀 (HFIP) </li><li> 낮은 결합 폴리 프로필렌의 microcentrifuge 튜브 (1.5 ML) </li><li> 테플론 동작 정류장 (250 μl)와 GasTight 해밀턴 주사기 </li><li> Dimethylsulfoxide (DMSO) </li></ul><ol><li> 동결 건조된 Aβ 1-42은 펩타이드 병을 열 때 응축을 피하기 위해 30 분 실온에서 평형 수 있습니다. </li><li> 펌 후드 아래, 1 MM 솔루션과 몇 초 동안 와동 솔루션을 얻기 위해 추운 얼음 HFIP에서 Aβ 1-42 펩타이드를 다시 일시 중지합니다. </li><li> 테플론 플러그와 유리 GasTight 해밀턴 주사기를 사용하여 신속하게 동일하게 세 폴리 프로필렌의 튜브에 Aβ 1-42 / HFIP 솔루션을 분할하고 튜브를 밀봉. </li><li> 튜브는 다음 Aβ monomerization 수 있도록 2 시간 incubated 수 있습니다. 다음 튜브를 열고 맑은 펩타이드 영화는 유리병의 하단에 관찰된 때까지 SpeedVac 원심 분리기 (800g, 실온)을 사용하여 진공에서 Aβ 1-42 / HFIP 솔루션을 집중. (최대 25 ° C) 펩타이드 저하를 피하기 위해 Speed​​Vac 원심 내부 신중하게 온도를 확인합니다. </li><li> 튜브를 밀봉하고 -80에서 aliquots를 저장 ° C. Aβ 영화는 6 개월 동안 저장할 수 있습니다. </li><li> 5 MM Aβ 1-42까지 농도를 얻을 DMSO를 추가하여 한 영화를 다시 일시 중지합니다. 다시 정지 완성하기 위해 10 분 동안 물 목욕으로 Sonicate. </li><li> 폴리 프로필렌의 튜브로 나누어지는이 5mM Aβ 1-42 솔루션입니다. 모든 튜브를 밀봉하고 -20 ° C.에 그들을 저장 Aliquots는 한 번만 해동해야합니다. 냉동실 내부에 물이 응축주의. 솔루션에서 펩티드 아주 쉽게 저하. </li></ol> Oligomerization 시작하기 전에 준비했습니다 : <ul><li> Aβ 1-42 / DMSO (5mM) </li><li> 멸균 인산 버퍼 </li><li> 낮은 결합 폴리 프로필렌의 microcentrifuge 튜브 (1.5 ML) </li><li> 낮은 결합 폴리 프로필렌의 원심 분리기 튜브 (50 ML) </li></ul><ol><li> 얻은 5mM Aβ 1-42 / 멸균 인산 버퍼 (최대 100 μl)와 DMSO의 나누어지는을 희석. </li><li> 30 초 동안 소용돌이 그리고 4에서 12 시간 동안 품어 ° C </li></ol> Hippocampal 슬라이스 처리 hippocampal 조각 치료 때, 비커, 재관류 튜빙 표면 및 녹음 챔버에 비 특정 펩타이드의 부착을 방지하는 것이 중요합니다. 낮은 결합 폴리 프로필렌 튜빙과 용기 우선적으로 사용합니다. 유리 또는 플라스틱 - 도자기 일반의 사용을 피하십시오. 재관류 미디어는 혈청 또는 알부민 무료되어야합니다. Aβ 재관류 시작하기 전에 준비했습니다 : <ul><li> Oligomerized의 Aβ 1-42 </li><li> 레코딩 버퍼 (㎜의 : 124 NaCl, 4.4 KCl, 1 나 2 HPO 4, 25 NaHCO 3, 2 CaCl 2, 2 MgCl 2, 10 포도당) </li><li> 인터페이스 챔버로 설정 Electrophysiological 기록 </li><li> 최소 90 분간 사후 해부를위한 산소 기록 버퍼에 incubated 트랜스 hippocampal 조각 (T = 29 ° C, 지속적인 재관류 율은 = 2 ML / 분) </li></ul><ol><li> 바로 전에 실험, 사전 산소 기록 버퍼와 폴리 프로필렌 튜브에서 해당 작업 농도 (200 NM 이상)에 oligomerized의 Aβ 1-42 나누어지는을 희석. </li><li> 시냅스 소성의 유도 전에 조각 20 분 동안 Aβ 1-42 솔루션을 Perfuse. </li></ol> 소성의 유도 및 후속 <ol><li> 일반적으로 시냅스 소성의 가장 널리 사용되는 모델은 LTP입니다. 이것은 해마에 주어진 버렸네의 파상풍의 자극을 통해 유도하실 수 있습니다. 우리는 샤퍼의 부수적인 전망과 지층의 radiatum의 CA1 피라미드 뉴런 사이의 시냅스에서 현장 응답을 기록합니다. 우리 파상풍의 자극을 15 초 간격으로 구분된 세 열차를 포함하는 세타 버스트 자극으로 구성되어 있습니다. 각 열차는 각 버스트은 100 Hz에서에서 5 펄스를 수반 5 Hz에서 10 파열로 구성되어 있습니다. oligomerized Aβ의 재관류가 완료되어 즉시 파상풍의 자극을 적용합니다. </li><li> 파상풍의 자극은 정상적인 녹음 버퍼와 재관류로 다시 전환 직후. 실험 기간에 걸쳐 지속적인 관류 속도, 기록 챔버 온도와 적절한 산소를 유지합니다. </li></ol> 대표 결과 및 가능한 문제 스타인 동부 표준시 콜의 전통적인 프로토콜입니다. 1에 따라 oligomerized Aβ는 Aβ 단량체 및 다양한 다른 크기의 oligomers를 생성S (이량체, trimer, 등) oligomerized의 재관류 제어 조각에 비해 Aβ - 취급 조각 감소 LTP에 Aβ 1-42 연결됩니다. 제어 조각에 LTP 가치가있는 동안 3~5개월 된 C57BL/6J 남성 마우스가 사용하는 우리의 실험 조건에서 LTP 200 NM에서 Aβ - 처리 슬라이스는 파상풍 자극 이후 두 시간에 기준 값을 평균 1백50%에 baseline2의 평균 200~250%에. 어떤 경우에는 Aβ와 치료 hippocampal LTP을 줄이기 위해 실패할 수 있습니다. 발생할 수있는 몇 가지 중요한 오류가 Aβ 농도와 불완전 oligomerization 동안 건조 과도한 필름을 포함합니다. hippocampal 조각의 Aβ 재관류는 우려 때문에 플라스틱이 아닌 특정 부착하는 경향이있다 솔루션 Aβ 펩타이드의 물리 특성의 또 다른 원천이 될 수 있습니다. 이러한 문제의 해결은 아래 설명되어 있습니다.

<ol>
	<li>Stine, W. B., Dahlgren, K. N., Krafft, G. A., LaDu, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+conditions+for+amyloid-beta+peptide+oligomerization+and+fibrillogenesis.">In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.</a> J Biol Chem. 278, 11612-11622 (2003).</li><li>Vitolo, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+-peptide+inhibition+of+the+PKA/CREB+pathway+and+long-term+potentiation:+reversibility+by+drugs+that+enhance+cAMP+signaling.">Amyloid beta -peptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling.</a> Proc Natl Acad Sci U S A. 99, 13217-13221 (2002).</li><li>Puzzo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picomolar+amyloid-beta+positively+modulates+synaptic+plasticity+and+memory+in+hippocampus.">Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus.</a> J Neurosci. 28, 14537-14545 (2008).</li><li>Lewczuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sample+collection+tubes+on+cerebrospinal+fluid+concentrations+of+tau+proteins+and+amyloid+beta+peptides.">Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid beta peptides.</a> Clin Chem. 52, 332-334 (2006).</li><li>Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+synaptic+dysfunction+in+Alzheimer's+disease.">Mechanisms of synaptic dysfunction in Alzheimer's disease.</a> Histol Histopathol. 10, 509-519 (1995).</li><li>Cullen, W. K., Suh, Y. H., Anwyl, R., Rowan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+of+LTP+in+rat+hippocampus+in+vivo+by+beta-amyloid+precursor+protein+fragments.">Block of LTP in rat hippocampus in vivo by beta-amyloid precursor protein fragments.</a> Neuroreport. 8, 3213-3217 (1997).</li><li>Itoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+of+long-term+potentiation+in+hippocampal+slices+of+beta-amyloid-infused+rats.">Impairments of long-term potentiation in hippocampal slices of beta-amyloid-infused rats.</a> Eur J Pharmacol. 382, 167-175 (1999).</li><li>Walsh, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+secreted+oligomers+of+amyloid+beta+protein+potently+inhibit+hippocampal+long-term+potentiation+in+vivo.">Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.</a> Nature. 416, 535-539 (2002).</li><li>Lambert, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nonfibrillar+ligands+derived+from+Abeta1-42+are+potent+central+nervous+system+neurotoxins.">nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.</a> Proc Natl Acad Sci U S A. 95, 6448-6453 (1998).</li><li>Gong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+hydrolase+Uch-L1+rescues+beta-amyloid-induced+decreases+in+synaptic+function+and+contextual+memory.">Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory.</a> Cell. 126, 775-788 (2006).</li><li>Trinchese, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+calpains+improves+memory+and+synaptic+transmission+in+a+mouse+model+of+Alzheimer+disease.">Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.</a> J Clin Invest. 118, 2796-2807 (2008).</li></ol>

동물 실험은 컬럼비아 대학 IACUC에 의해 명시된 지침과 규정에 따라 수행되었다.

위에서 설명한 바와 같이, oligomeric Aβ의 준비에 몇 가지 문제가 손상되지 않은 LTP가 발생할 수 있습니다. Aβ 열화 또는 Aβ oligomerization의 부족이 발생했을 수 있는지 여부를 평가하는 한 가지 방법은 트리스 - Tricine 페이지 웨스턴 / 모래 바닥 분석 및 분석 ultracentrifugation (이 프로토콜에서 설명하지 않은)를 사용하여 Aβ 준비를 평가하는 것입니다. 서양 모래 바닥 샘플 non-denaturing/non-reducing 조건 하?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>A&beta; 1-42 (lyophilized) </td>
<td>&nbsp;</td>
<td>American Peptide Co.</td>
<td>62-0-80</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>52517</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Biotium</td>
<td>90082</td>
<td>&nbsp;</td>
<tr>
<td>50mL Polypropylene Centrifuge Tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>05-538-67</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml MaxyClear microtubes Maximum recovery</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT-150-L-C</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-Buffered Saline pH 7.4</td>
<td>&nbsp;</td>
<td>Invitrogen-Gibco</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>GASTIGHT Syringes 250 &mu;L</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>1725</td>
<td>&nbsp;</td>
<tr>
<td>Bath sonicator</td>
<td>&nbsp;</td>
<td>Branson</td>
<td>3510</td>
<td>&nbsp;</td>
<tr>
<td>Savant SpeedVac Concentrator System</td>
<td>&nbsp;</td>
<td>Various distributors</td>
<td>SC 110A</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation of oligomeric &beta;-amyloid1-42 and induction of synaptic plasticity impairment on hippocampal slices

시냅스 연결의 장애가 알츠하이머 병 (AD)의 초기 단계에서 발생하는 미묘한 amnesic 변경 사항을 기초로 가능성이 높습니다. β - 아밀로이드 (Aβ), AD 높은 금액에서 생산되는 펩티드는 세포가 학습과 기억의 상관 관계를 장기 Potentiation (LTP)을 감소시키는 것으로 알려져 있습니다. 사실, Aβ에 의한 LTP 장애는 AD 모델과 같은 신경 손상을 완화하거나 되돌리는 능력 심사 약물에 대한 신경 장애를 공부에 유용한 실험 패러다임이다. 연구 Aβ는 oligomeric 양식을 통해 우선적으로 LTP 장애를 생산하는 것으로 나타났습니다. 여기 oligomerized 합성의 재관류에 의해 급성 hippocampal 조각에 Aβ1 - 42 펩타이드를 혼란 LTP에 대한 자세한 프로토콜을 제공합니다. 이 비디오에서는, 우리는 oligomeric Aβ의 준비를위한 단계별 절차를 설명합니다<sub&gt; 1-42</sub&gt;. 그렇다면, 우리는 LTP가 oligomerized Aβ에 노출 hippocampal 조각 축소되는 개별 실험을 수행<sub&gt; 1-42</sub&gt; 노 Aβ 제어 실험에서 조각에 비해<sub&gt; 1-42</sub&gt; 노출이 발생했다.

Watch this Scientific Journal Video about Preparation of Oligomeric Î²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices at JoVE.com

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

알츠하이머병 중 하나는 기능은 Aβ의 상승입니다<sub&gt; 1-42</sub&gt; 펩타이드. 여기 합성 Aβ를 준비를위한 프로토콜을 제공합니다<sub&gt; 1-42</subhippocampal 장기 Potentiation을 impairs&gt; oligomers는, 세포의 메모리 상관 관계. 이 절차는 Aβ 유발 병리학 및 약물 검사의 메커니즘을 조사하는 데 유용합니다.

β - 아밀로이드 Oligomeric의 준비 1-42</subHippocampal 조각에 시냅틱 소성 장애의>와 유도

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

preparation-oligomeric-amyloid1-42-induction-synaptic-plasticity

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Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

23.0K 조회수.  Columbia University. 알츠하이머병 중 하나는 기능은 Aβ의 상승입니다 1-42 펩타이드. 여기 합성 Aβ를 준비를위한 프로토콜을 제공합니다 1-42 oligomers는, 세포의 메모리 상관 관계. 이 절차는 Aβ 유발 병리학 및 약물 검사의 메커니즘을 조사하는 데 유용합니다.

비디오: Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

노화와 아밀로이드 병리 동안 시냅틱 얼터 레이션의 연구에 대한 고양이와 트렌스 제닉 마우스의 급성 Hippocampal 조각의 준비

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

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신경과학

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

쥐 뇌 조각에서 뉴런의 Vibrodissociation는 시냅틱 전송 및 이미지 Presynaptic 터미널 공부

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

교양 뉴런의 막 단백질의 측면 보급과 Exocytosis은 형광 복구 및 형광 손실 Photobleaching를 사용하여 평가

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Hippocampal 슬라이스 문화의 시냅틱 단백질의 포스트 퍼가기 Immunogold 라벨링

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

장기 상승 작용으로 매우 안정하고 재현성 기록을위한 향상된 준비 및 해마 마우스 조각의 보존

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

마우스 해마에 올리고머 아밀로이드 - 베타의 정위 주입

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

설치류에서 급성 해마 조각을 사용하여 시냅스 태그 / 캡처 및 크로스 캡처 조사

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

교양된 Hippocampal 신경에 시 냅 스 소포 Endocytosis를 측정

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

급성 Hippocampal 조각에 작은 볼륨 재활용-, 관류-침수 형 챔버 시스템 유지에 시 냅 스가 소성을 기록

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

시 냅 스 밀도 Hippocampal 설치류 두뇌 분할 영역에서의 평가

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

β - 아밀로이드 Oligomeric의 준비 1-42와 유도

β - 아밀로이드 Oligomeric의 준비_{1-42와 유도}