This work was funded by the DFG (SFB 779 TPB8, Kr1879/3-1 MRK), DIP grant (MRK), EU FP7 MC-ITN NPlast (MRK), Center for Behavioral Brain Sciences (CBBS, Sahsen-Anhalt), (AK and SB), MM is a recipient of European Molecular Biology Organization (EMBO) Long-Term Fellowship (EMBO ALTF 884-2011) and Marie-Curie IEF.

성인 쥐의 뇌에서 급성 해마 조각의 1 준비 <ol><li> 이소 플루 란 쥐를 마취. 주의 : 폐쇄 exicator를 사용하여 절차를 수행, 이소 플루 란을 흡입하지 않습니다. 동물이 완전히 마취되어 있는지 확인합니다. </li><li> 신속하게 쥐의 목을 벨 뇌를 분리하고, precarbonated (95 %, O2 / 5 % CO 2 가스 혼합물) 얼음처럼 차가운 밀리미터의 Gey의 솔루션 (구성에 젖어 : (130)의 NaCl, 4.9의 KCl, 1.5 염화칼슘 2 · 2H 2 O를 0.3 황산 4 · 2H 2 O, 11 MgCl2를 · 6H 2 O, 0.23 KH 2 PO 4, 0.8 나 2 HPO 4 · 7H 2 O, 5 포도당 · H 2 O, 25 HEPES, 22 탄산 수소 나트륨, 산도 7.32) 30 분 1,2,10,16합니다. </li><li> entorhinal 피질의 소뇌 부분을 제거합니다. 다음의 내측 표면에 각각의 반구를 배치, 중간 시상 컷 대뇌 피질 반구를 분리합니다. 그 후 확인각 반구 (13, 14)의 지느러미 가장자리를 따라 50 ~ 70 ° 컷 (50 ~ 70 °의 횡 방향). </li><li> 절편 시스템의 슬라이스 플랫폼에서 갓 잘라 표면과 각 반구를 붙인다. 이 플랫폼은 precarbogenated 얼음처럼 차가운 Gey의 솔루션이 적용되어야한다. </li><li> z 축 진동을 최소화하기 위해 조정 된 vibratome를 사용하여 다른 쪽면 후부하기 위해 전방에서 350 μm의 50 ~ 70 ° 횡단면을 잘라. 해마 형성 subicular 및 entorhinal 피질뿐만 아니라 해마에있는 dorsolateral을 피질 13,14 실험에 사용 슬라이스의 일부가 될 것이다. </li><li> U 모양 및 침수 형 인큐베이터에 해마 슬라이스를 전송하고 carbogenated 인공 뇌척수액 (ACSF (단위 : mm) 포함 32 ° C에서 적어도 2 시간 동안 품어 : 2H 2 O, 1.5 황산 · 110 염화나트륨, 2.5의 KCl, 2.5 염화칼슘이 4 · 2H 2 O, 1.24 KH 2 PO 4, 10 포도당 · H 2O, 27.4 탄산 수소 나트륨, 산도 7.3) 1,2,10,16. </li></ol> 2 전극의 위치, 기준 기록 및 LTP의 유도 <ol><li> 현미경 장착 침수 타입의 기록 챔버에 해마 슬라이스를 전송합니다. 32 ° C에서 최소 30 분 동안 carbogenated ACSF로 (6-7 ml / 분)를 관류. </li><li> (팁 저항 MΩ 3-5 인) ACSF 가득 유리 모세관 미세 전극을 준비합니다. </li><li> 자극과 fEPSP의 CA1 지층 radiatum 기록 1,2,10 (그림 2)에서 CA1 샤퍼 담보 섬유에 ACSF 가득 유리 미세 전극을 배치합니다. 전극 사이의 거리는 약 300 μM이어야한다. </li><li> 3-4 V.의 범위에서 이상성 직사각형 전류 펄스 (200 밀리 초 / 극성)와 샤퍼 담보 섬유의 자극에 의​​해 필드 흥분성 시냅스 후 전위 (fEPSPs)를 보여주고 있습니다 </li><li> INP을 측정함으로써 최대 자극 검사를 수행UT 출력 관계가 얻어 fEPSP 최대 슬로프 값의 40 %의 자극 강도를 정의하고 실험 내내 일정하게 유지. </li><li> 최대 자극 검사 후 최소 15 분 동안베이스 라인 녹음을 시작합니다. 실험을하는 동안 모든 분을 자극 테스트 응답을 측정한다. 저주파 자극과베이스 라인 녹음을 수행합니다. </li><li> 적어도 30 분 동안 기준선을 기록합니다. </li><li> 늦은 LTP 유도를 들어 고주파에게 5 분 intertrain 간격으로 100 Hz에서 세 1 초 자극 열차로 구성된 100 Hz에서 tetanization을 적용합니다. CA1 지역 5 μM의 bicuculline 내 자극의 필드를 증가시키기는 tetanization 전에 2 분 세척 할 수 있으며, 즉시 마지막 tetanization 후에 세척해야한다. </li></ol> LTP와 스냅 동결 유도 후 조각의 3 컬렉션 <ol><li> 정지 LTP는 2 분이나 늦게 LTP를 유도 파상풍의 자극 세 열차 후 30 분을 기록. </li><l난&gt; 전극을 제거하고 신속하게 드라이 아이스에 배치 precold 금속 플랫폼에 슬라이스를 전송합니다. U 모양의 인큐베이터에 보관 제어 슬라이스에 대해이 절차를 반복합니다. 주의 : 장갑 드라이 아이스와 함께 작업하는 동안. </li><li> -80 ° C에서 1.5 ML 튜브 및 저장소에있는 각 조각을 수집합니다. </li></ol> 해마의 CA1 지역에서 핵 농축 분획의 4 격리 <ol><li> -80 ° C에서 냉동 슬라이스로 1.5 ㎖의 에펜 도르프 튜브를 타고 얼음에 보관하십시오. </li><li> 추가 튜브에 신선한 차가운 TBS 버퍼를 포함하는 단백질 분해 효소 (PI) 및 포스 파타 아제 (PS) 억제제, 0.5 ml의. 2 ~ 3 분 동안 품어 실체에 전송합니다. 주의 : 보호 장갑과 PI와 PS 작업하는 동안 실험실 코트. </li><li> 두 바늘을 사용하여 해마의 CA1 지층 pyramidale 영역을 해부하다. 실체와 CA1 영역의 절단 초 바늘 아래 슬라이스를 유지하기위한 하나의 바늘을 사용한다. </li><li> 해부 CA를 수집50 ㎕의 용해 완충액을 함유하는 새로운 1.5 ㎖의 튜브에 (각 그룹에 대해) 5 조각에서 한 지역 (mm 단위 함유 저장성 용해 완충액 1x에서 10 HEPES를, 1.5 MgCl2를, 10의 KCl, PI 및 PS으로 pH 7.9). 소재의이 금액은 2 ~ 3 immunoblots을 실행하기에 충분한 있습니다. </li><li> 아래주의 피펫 위로에 의해 수집 된 조직을 균질화. 200 μL 피펫을 사용합니다. 세포가 팽창 할 수 있도록 5-7 분 동안 얼음에 해물을 품어. </li><li> , 해물의 2 μl를 가지고 현미경 슬라이드를 삭제하고 밝은 필드 현미경으로 세포의 부종을 시각화. 세포의 적절한 팽창으로 핵은 원형 그대로 구조를 나타납니다. </li><li> &#39;균질 부분&#39;등 신선한 1.5 ML 튜브에 샘플의 20 μl를 수집합니다. 배 SDS 샘플 버퍼를 변성의 8 μl를 추가합니다. </li><li> 1 분 동안 11,000 rpm에서 원심 분리 잔류 해물. </li><li> 조심스럽게, 그럴 상단 ( &#39;세포질 부분&#39;)에서 상층 액을 수집ansfer 새로운 튜브에 넣고 4 배의 샘플 버퍼의 20 μl를 추가합니다. </li><li> 저장성 버퍼의 60 μL에 펠렛을 재현 탁하고 배 샘플 버퍼의 20 μl를 추가합니다. 이 분획을 &#39;핵 농축 분획&#39;라 칭한다. </li><li> 균질, 세포질 및 핵 농축 분획 또는 -80 나중에 면역 블로에 대한 C ° -20 ° C에서 저장 될 수있다. </li></ol> Synapto 핵 단백질에 대한 5 반 정량적 Immunoblotting 방법 <ol><li> 샘플을 Defreeze 95 ° C에서 5 분간을 끓인다. </li><li> 각 부분에서 5 μl를 타고 아미 검정 시험 또는 BCA 시험에 의해 단백질 농도를 결정한다. </li><li> SDS-PAGE에 균질에서 단백질 시료의 같은 양의 세포질과 핵 농축 분획을로드합니다. 제어 및 LTP의 조각에서 샘플을 직접 비교를 위해 동일한 겔에 배치해야합니다. </li><li> 표준 서구 블로 팅 절차를 수행 (젖은 전송 권장). </li><li> t으로 막을 프로브그는 선택의 항체의. 특정한 뉴런 enolase2 (NSE2)과 핵 마커 - - NeuN 및 로딩 대조군으로서 액틴 항체이어서 동일 롯 (또는 병렬로 실행 동일한 샘플) 세포질 마커를 탐색 할 수있다. </li></ol> (6) 데이터 분석 <ol><li> LTP 유도의 효율 Clampfit 소프트웨어에 의해 분석 될 수있다. 기준선 기록의 평균 기울기 tetanization 후 경사면 양방향 ANOVA, P를 사용하여 비교 하​​였다 &lt;0.05는 유의 한 차이로 간주 하였다. fEPSP 기울기 값은 SEM 평균 ±로 다이어그램에 묘사 된 </li><li> immunoblots의 정량, 방사 법적 필름 중 하나를 스캔 Licor 시스템 ImageJ에 또는 형광 단백질 밴드에 의한 대역의 집적 밀도를 분석한다. 면역 밴드의 값은로드 및 블로 팅 제어를 정상화해야합니다. 컨트롤의 비교 및​​ LTP 그룹의 경우 nonparametrical 맨 - 휘트니 U 테스트를 사용할 수 있습니다. </li></ol><table border="0"cellpadding이 = &quot;0&quot;cellspacing = &quot;0&quot;&gt; <tbody><tr><td> 버퍼의 이름 </td><td> 시약 </td><td> 농도 (㎜) </td><td> 금액 </td><td> 댓글 / 설명 </td></tr><tr><td> Gey의 솔루션 (산도 : 7.4 ~ 7.3) </td><td> 염화나트륨 </td><td> 130 </td><td> 7.6 g </td><td> 멸균 여과 </td></tr><tr><td> 1000 ML </td><td> 의 KCl </td><td> 4.9 </td><td> 0.37 g </td><td></td></tr><tr><td></td><td> 염화칼슘 2 · 2H 2 O </td><td> 1.5 </td><td> 0.22 g </td><td></td></tr><tr><td></td><td> 황산 4 · 2H 2 O </td><td> 0.3 </td><td> 0.0739 g </td><td></td></tr><tr><td></td><td> MgCl2를 · 6H 2 O </td><td> 11 </td><td> 2.24 g </td><td></td></tr><tr><td></td><td> KH 2 PO 4 </td><td> 0.23 </td><td> 0.0313 g </td><td></td></tr><tr><td></td><td> 나 2 HPO 4 · 7H 2 O </td><td> 0.8 </td><td> 0.2145 g </td><td></td></tr><tr><td></td><td> 포도당 · H 2 O </td><td> 5 </td><td> 0.9909 g </td><td></td></tr><tr><td></td><td> HEPES </td><td> 25 </td><td> 5.96 g </td><td></td></tr><tr><td></td><td> 탄산 수소 나트륨 </td><td> 22 </td><td> 1.85 g </td><td></td></tr><tr><td> ACSF (산도 : 7.4 ~ 7.3) </td><td> 염화나트륨 </td><td> (110) </td><td> 6.428 g </td><td> 멸균 여과 </td></tr><tr><td> 1000 ML </td><td> 의 KCl </td><td> 2.5 </td><td> 0.1865 g </td><td></td></tr><tr><td></td><td> 염화칼슘 2 · 2H 2 O </td><td> 2.5 </td><td> 0.368 g </td><td></td></tr><tr><td></td><td> 황산 4 · 2H 2 O </td><td> 1.5 </td><td> 0.370 g </tD&gt; <td></td></tr><tr><td></td><td> KH 2 PO 4 </td><td> 1.24 </td><td> 0.169 g </td><td></td></tr><tr><td></td><td> 포도당 · H 2 O </td><td> 10 </td><td> 1.9817 g </td><td></td></tr><tr><td></td><td> 탄산 수소 나트륨 </td><td> 27.4 </td><td> 2.3 g </td><td></td></tr><tr><td> 1 배 저장성 완충액 (pH : 7.9) </td><td> HEPES </td><td> 10 </td><td> 0.23 g </td><td></td></tr><tr><td> 100 ml의 </td><td> MgCl2를 · 6H 2 O </td><td> 1.5 </td><td> 0.0304 g </td><td></td></tr><tr><td></td><td> 의 KCl </td><td> 10 </td><td> 0.07455 g </td><td></td></tr></tbody></table> 표 1 버퍼.

<ol>
	<li>Behnisch, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+translocation+of+Jacob+in+hippocampal+neurons+after+stimuli+inducing+long-term+potentiation+but+not+long-term+depression.">Nuclear translocation of Jacob in hippocampal neurons after stimuli inducing long-term potentiation but not long-term depression.</a> PLoS One. 6 (2), e17276 (2011).</li><li>Cai, F., Frey, J. U., Sanna, P. P., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+degradation+by+the+proteasome+is+required+for+synaptic+tagging+and+the+heterosynaptic+stabilization+of+hippocampal+late-phase+long-term+potentiation.">Protein degradation by the proteasome is required for synaptic tagging and the heterosynaptic stabilization of hippocampal late-phase long-term potentiation.</a> Neuroscience. 169 (4), 1520-1526 (2010).</li><li>Chapman, C. A., Perez, Y., Lacaille, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+GABA(A)+inhibition+on+the+expression+of+long-term+potentiation+in+CA1+pyramidal+cells+are+dependent+on+tetanization+parameters.">Effects of GABA(A) inhibition on the expression of long-term potentiation in CA1 pyramidal cells are dependent on tetanization parameters.</a> Hippocampus. 8 (3), 289-298 (1998).</li><li>Ch'ng, T. H., Uzgil, B., Lin, P., Avliyakulov, N. K., O'Dell, T. J., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+transport+of+the+transcriptional+coactivator+CRTC1+from+synapse+to+nucleus.">Activity-dependent transport of the transcriptional coactivator CRTC1 from synapse to nucleus.</a> Cell. 150 (1), 207-221 (2012).</li><li>Dieterich, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caldendrin-Jacob:+a+protein+liaison+that+couples+NMDA+receptor+signalling+to+the+nucleus.">Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus.</a> PLoS Biol. 6 (2), e34 (2008).</li><li>Fainzilber, M., Budnik, V., Segal, R. A., Kreutz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+synapse+to+nucleus+and+back+again--communication+over+distance+within+neurons.">From synapse to nucleus and back again--communication over distance within neurons.</a> J. Neurosci. 31 (45), 16045-16048 (2011).</li><li>Hardingham, G. E., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+versus+extrasynaptic+NMDA+receptor+signalling:+implications+for+neurodegenerative+disorders.">Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders.</a> Nat. Rev. Neurosci. 11 (10), 682-696 (2010).</li><li>Ivanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+role+of+synaptic+and+extrasynaptic+NMDA+receptors+in+regulation+of+the+extracellular+signal-regulated+kinases+(ERK)+activity+in+cultured+rat+hippocampal+neurons.">Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons.</a> J. Physiol. 57, 789-798 (2006).</li><li>Jordan, B. A., Kreutz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+protein+shuttling:+the+direct+route+in+synapse-to-nucleus+signaling.">Nucleocytoplasmic protein shuttling: the direct route in synapse-to-nucleus signaling.</a> Trends Neurosci. 32 (7), 392-401 (2009).</li><li>Karpova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding+and+transducing+the+synaptic+or+extrasynaptic+origin+of+NMDA+receptor+signals+to+the+nucleus.">Encoding and transducing the synaptic or extrasynaptic origin of NMDA receptor signals to the nucleus.</a> Cell. 152 (5), 1119-1133 (2013).</li><li>Kim, M. J., Dunah, A. W., Wang, Y. T., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+NR2A-+and+NR2B-containing+NMDA+receptors+in+Ras-ERK+signaling+and+AMPA+receptor+trafficking.">Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking.</a> Neuron. 46, 745-760 (2005).</li><li>Lai, K. O., Zhao, Y., Ch'ng, T. H., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importin-mediated+retrograde+transport+of+CREB2+from+distal+processes+to+the+nucleus+in+neurons.">Importin-mediated retrograde transport of CREB2 from distal processes to the nucleus in neurons.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (44), 17175-17180 (2008).</li><li>Leutgeb, J. K., Frey, J. U., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTP+in+cultured+hippocampal-entorhinal+cortex+slices+from+young+adult+(P25-30)+rats.">LTP in cultured hippocampal-entorhinal cortex slices from young adult (P25-30) rats.</a> J. Neurosci. Methods. 130 (1), 19-32 (2003).</li><li>Leutgeb, J. K., Frey, J. U., Behnisch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+activity-dependent+cyclic+AMP-responsive+element-binding+protein+phosphorylation+during+long-lasting+long-term+potentiation+in+area+CA1+of+mature+rat+hippocampal-organotypic+cultures.">Single cell analysis of activity-dependent cyclic AMP-responsive element-binding protein phosphorylation during long-lasting long-term potentiation in area CA1 of mature rat hippocampal-organotypic cultures.</a> Neuroscience. 131 (3), 601-610 (2005).</li><li>Perlson, E., Hanz, S., Ben-Yaakov, K., Segal-Ruder, Y., Seger, R., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vimentin-dependent+spatial+translocation+of+an+activated+MAP+kinase+in+injured+nerve.">Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve.</a> Neuron. 45 (5), 715-726 (2005).</li><li>Xiang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+maintenance+of+mature+hippocampal+slices+in+vitro.">Long-term maintenance of mature hippocampal slices in vitro.</a> J. Neurosci. Methods. 98 (2), 145-154 (2000).</li><li>Zhao, M., Adams, J. P., Dudek, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern-dependent+role+of+NMDA+receptors+in+actin+potential+generation:+consequences+on+extracellular+signal-regulated+kinase+activation.">Pattern-dependent role of NMDA receptors in actin potential generation: consequences on extracellular signal-regulated kinase activation.</a> J. Neurosci. 25 (30), 7032-7039 (2005).</li></ol>

The authors have nothing to disclose.

위의 프로토콜에 설명 된 단계는, 해마 급성 젊은이나 성인 쥐에서 슬라이스 준비 슬라이스의 자극 영역을 유도하고 기록 LTP가 신속하게 해부하고, 활동에 따라 단백질 역학을 공부 핵 농축 분획을 준비하는 방법에 대한 지침을 제공합니다. 이러한 접근 방식은 서로 독립적으로 사용되는 몇 가지 상이한 방법의 조합으로부터 도출한다. 우리는 워크 플로우를 최적화 LTP의 유도에 따라 단백질의 세...

시냅틱 N-메틸-D-아스파 테이트는 수용체 (NMDARs)는 시냅스 가소성 및 세포 생존이 신경 퇴행 및 세포 사멸을 유발할 수 extrasynaptic NMDARs 반면 시그널링의 활성화에 결정적인 역할을한다. 이러한 변화는 엄격하게 제어 / 규제 활동에 따라 유전자 발현에 의존하므로 활성화 시냅스 또는 수상 돌기 및 핵 7 사이의 지속적인 통신을 필요로한다. MAP는 ERK1 / 2 신호를 시냅스 NMDARs의 다운 스트림 이펙터하고 extrasynaptic NMDAR를 통해 시그널링 없거나 ERK1 / 2 활성 8,11에 대한 억제 효과가있는 반면, NMDAR 활성화에 의한 유전자 발현에 관여하지 않음을 키나아제. 원위 수상 돌기와 핵 사이의 셔틀로 표시되었습니다 단백질의 여러 가지가 있습니다. 이들 단백질의 대부분은 핵 이행 시그널을 포함하고 적극적으로 핵 6,9 네인과 임포 의존적으로 microtubuli을 따라 수송된다. InterestinglY, 특정 시냅스 자극에 대한 응답으로 핵이 사자의 일부는 교통. 예를 들어, 사이 클릭 AMP 반응 요소 결합 단백질이 (CREB2)의 역행 수송 화학 LTD 아니라 LTP (12)에 의해 유도된다. 지역화 NMDAR 종속 시냅스 자극 장기 해마 가소성 4에서 포함되는 핵 전좌 프로세스로 CREB 조절 전사 보조 활성화 (CRTC1)를 구동한다. 그것은 최근에 단백질 메신저 야곱이 모두 후 핵 시냅스와 extrasynaptic NMDAR 활성화에 옭 및 CREB 따라 유전자 전사 5를 조절 것으로 나타났다. 신호의 시냅스 또는 extrasynaptic 원점은 야곱의 번역 후 변형에 인코딩된다. 시냅스 활동이 주​​ 해마 문화의 핵 이후의 전위에 대한 필수입니다 위치 180 (pJacobS 180)에서 중요한 세린에서 야곱의 ERK1 / 2 의존 인산화를 유도한다. 또한, 전N CA1의 샤퍼 담보 LTP 후 핵 급성 해마 조각 pJacobS 180 옭의 뉴런하지만은 1,10 LTD. pS180 야곱은 가소성 관련 유전자의 발현 증가로 연결이 유전자 발현은 시냅스 기능에 피드백. extrasynaptic NMDARs의 활성화가 Ser180 인산화되지 않고 원인이 핵에 다른 단백질 복합체와 연관 될 수 있습니다 후 핵 옭 대조적으로, 야곱에서 &#39;CREB는 차단&#39;와 연접 (10)의 후퇴. synapto 핵 단백질 메신저의 핵 수입에 출판 된 대부분의 연구는 해리의 연결을 기본 문화에서 수행되고있다. 신경 세포의 연결 및 기능이 매우 잘 보존 된 곳 따라서 이러한 연구 결과는 해마 조각을 사용하여 생리 학적 관련성 조건에서 재현 할 수 있는지 흥미로운 일이 될 것이다. 여기에서 우리는 LTP 드를 평가하기위한 최적화 된 프로토콜을 제시면역 블로에 의해 단백질 메신저의 핵 전좌 매달린. 이 방법은 조 핵 분획 단백질의 활성에 의존 인산화 분석에 적합하다. 구체적으로는, 현재 프로토콜은 급성 CA1 해마 슬라이스, 유도, 및 LTP의 기록의 준비를 포함한다. 다음에, CA1 영역 현미경 자극 영역을 분리 해부된다. 우리는 결합 조 및 동료 (17)에 의해 도입 된 변경 CellLytic 핵 추출 키트에서 제공하는 핵 분리를위한 프로토콜을 수정했습니다. 최적화 과정은 세포 부종과 핵의 분리를 허용 저장성 버퍼 해부 CA1 영역의 용해를 포함한다. 세포 용해 및 핵의 형태를 현미경 검사에 의해 결정될 수있다. 핵 농축은 짧은 원심 분리 단계에 의해 달성된다. NeuN 및 NSE​​2 핵 또는 세포질 분획 특정 마커에 대한 항체로 면역 블 롯팅 분석을하는 것은이 방법은 고속으로 사용될 수 있음을 나타낸다재생 가능한 프로토콜은 이러한 세포 내 분수를 분리하고 단백질 인산화와 같은 매우 불안정한 번역 후 변형을 연구. 또한,이 방법은 해마 슬라이스 해부 CA1 영역으로부터 도출 작은 조직 샘플에 유리하고 해마 슬라이스에 면역을 조합하여 사용할 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Equipment</td>
		</tr>
		<tr>
			<td>CED 1401 AD/DA converter</td>
			<td>Cambridge Electronics Design, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/data_acquisition_system">Axon Digidata acquisition system</a></td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 10.0 Data acquisition and analysis software</td>
			<td>Axon&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope</td>
			<td>Leica TCS SP2&#65292;Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P-97 Standard microelectrode puller</td>
			<td>Sutter&#65292;USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.iciba.com/stereomicroscope">Stereomicroscope</a></td>
			<td>Leica</td>
			<td>Leica S4E, Germany</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica VT1000S, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated pulse stimulator</td>
			<td>Model2100, A-M systmes, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, HERAEUS, FRSCO17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE system</td>
			<td>BIORAD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>JULABO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field Microscope</td>
			<td>NIKON ECLIPSE TS100</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Electron Corporation, HERAEUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Luigs &#38; Neumann, SM-5, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting chamber and electric power supplier</td>
			<td>Hoefer Scientific Instruments, San Francisco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>Julabo, F12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific, Heraeus, FRESCO 17</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Submerged type Recording Chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-shape and submerged type incubator&#160;</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic culture dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Reagents</td>
		</tr>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.3957.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>ROTH</td>
			<td>Art.-Nr.6781.1</td>
			<td>&#8805;99.5%, p.a.,ACS,ISO</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.02382.0500</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgSO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>5886.05</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>AppliChem</td>
			<td>CAS-NO: 7791-18-6; EC-NO:2320946</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MERCK</td>
			<td>12034.025</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;2H2O</td>
			<td>MERCK</td>
			<td>1.06574.1000</td>
			<td>extra pure</td>
		</tr>
		<tr>
			<td>Glucose&#183;H2O</td>
			<td>ROTH</td>
			<td>Art.-Nr.6887.1</td>
			<td>for Molecularbiology</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>ROTH</td>
			<td>Art.-Nr.9105.4</td>
			<td>PUFFERAN, &#8805;99.5%, p.a.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>MERCK</td>
			<td>1.06329.1000</td>
			<td>pro analysi</td>
		</tr>
		<tr>
			<td>Protease inhibitor Coctail</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphostop</td>
			<td>ROCHE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bicuculline</td>
			<td>Tocris bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Isofluran</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Antibodies</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Species</td>
		</tr>
		<tr>
			<td>pJac-s180</td>
			<td>Biogenes/ purified</td>
			<td></td>
			<td>rabbit (dil. 1:100)</td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Milipore</td>
			<td>MAB377</td>
			<td>mouse (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>NSE</td>
			<td>Cell Signaling</td>
			<td>D20H2</td>
			<td>rabbit (dil. 1:1,000)</td>
		</tr>
		<tr>
			<td>beta-actin</td>
			<td>Sigma</td>
			<td>A-5441</td>
			<td>mouse (dil. 1:5,000)</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td>Species</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>DAKO</td>
			<td></td>
			<td>goat anti - mouse (1:5,000)</td>
		</tr>
		<tr>
			<td>IgG HRP Conjugated</td>
			<td>NEB</td>
			<td></td>
			<td>goat anti - rabbit (1:5,000)</td>
		</tr>
	</tbody>
</table>

isolation of ca1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins

학습 활동에 따라 단백질 발현, 세포 내 전위 또는 인산화는 시냅스 가소성의 기본 세포 메커니즘을 이해하는 것이 필수적이다. 장기 증강 (LTP) 및 급성 해마 조각에 의한 장기적인 우울증 (주)는 널리 학습과 기억의 세포 모델로 허용됩니다. 활동에 따라 단백질 역학을 시각화하는 라이브 세포 이미징 또는 면역 조직 화학 염색 방법을 사용하여 많은 연구가있다. 그러나 이러한 방법은 하나의 신경 세포에서 형광 표지 된 단백질의 면역 세포 또는 과발현에 대한 항체의 적합성에 의존하고 있습니다. 단백질의 면역 블로는 연구 결과의 독립적 인 확인을 제공하는 다른 방법이다. 개별 tetanized 해마 조각에서 세포 내 분수의 준비의 첫 번째 제한 요소는 재료의 낮은 금액입니다. 둘째로, 처리 절차는 중요 리터 심지어 매우 짧고 작은 조작 인해슬라이스를 iving하는 특정 신호 폭포의 활성화를 유도 할 수 있습니다. 여기에서 우리는 쥐의 뇌에서 급성 해마 슬라이스의 CA1 영역에서 충분한 순도의 핵 농축 부분의 충분한 양을 얻기 위해 최적화 된 워크 플로우를 설명합니다. 대표적인 예로서 우리는 synapto 핵 단백질 메신저의 ERK1 / 2의 인산화 형태 야곱이 적극적으로 LTP의 유도에 따라 핵에 옭 및 CA1 신경 세포에서 핵 농축 분획에서 검출 될 수 있다는 것을 보여줍니다.

우리는 이전에 synapto 핵 단백질 메신저 야곱이 LTP의 유도하지만 LTD 한 다음 핵에 축적 것으로 나타났습니다. 또한, 시냅스 자극 후 야곱의 전위는 Ser180 (그림 1)에서 MAPK ERK1 / 2의 활성화와 야곱의 인산화가 필요합니다. 인산화 야곱은 임포 의존적으로 핵에 옭 및 인산화 상태는 15 αinternexin 중간 필라멘트와 관련하여 (그림 1)에 의해 장기간에 걸쳐 보존 할 ...

Watch this Scientific Journal Video about Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins at JoVE.c

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

우리의 해마 CA1 영역과 슬라이스 tetanized 영역으로부터 핵 농축 분획의 분리 이후에 장기간 상승 작용의 유도에 대한 상세한 프로토콜을 제공한다. 이 방법은 활성보기 학습 및 기억의 세포 모델에 의존하는 핵 단백질 수입을 결정하기 위해 사용될 수있다.

해마 조각에서 CA1 핵 농축 분획의 분리는 Synapto 핵 메신저 단백질의 활동에 의존하는 핵 가져 오기를 공부합니다

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

isolation-ca1-nuclear-enriched-fractions-from-hippocampal-slices-to

Research

JoVE Journal

Neuroscience

12.0K 조회수.  Leibniz Institute for Neurobiology.  우리의 해마 CA1 영역과 슬라이스 tetanized 영역으로부터 핵 농축 분획의 분리 이후에 장기간 상승 작용의 유도에 대한 상세한 프로토콜을 제공한다. 이 방법은 활성보기 학습 및 기억의 세포 모델에 의존하는 핵 단백질 수입을 결정하기 위해 사용될 수있다. 

비디오: Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

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 ...

연구

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 ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

태아 쥐의 Hippocampal 신경 세포의 분리 및 문화

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

미토콘드리아 - 관련 ER의 멤브레인 (MAMs)와 Glycosphingolipid 강화 Microdomains (보석) : 마우스 뇌에서 절연

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

해마의 Organotypic 조각의 쌍으로 전체 셀 녹음

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...

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 ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

만성 실리콘 프로브 및 녹음 풍부한 디딜 방 아 장치에 Hippocampal 장소 세포의 이식

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

마 취 쥐에서 척수의 컬렉션에 대 한 개선된 방법

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

전기 생리학적 기록을 위한 급성 인간 해마 슬라이스의 준비

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...