The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

다음 프로토콜은 동물 관리의 캐나다 협의회의 가이드 라인을 준수하고 토론토 대학에서 의학 및 약학 동물 관리위원회의 교수진에 의해 승인되었습니다. 표 1에서 설명한 것과 같이 필요한 시약을 준비 <ol><li> 표 1에 설명 된 농도에서 모두 자당 솔루션, 버퍼에 단백질 분해 효소와 포스 파타 아제 (필요한 경우) 억제제를 추가하고 DDH 2 O. 중요 : 실험의 시작하기 전에 시약 및 장비를 모두 4 ° C에서 모든 단계를 수행하고 사전 멋진. 영구 마커 초 원심 분리기 튜브를 포함한 모든 튜브 레이블. </li></ol> 적절한 두뇌 지역의 2 해부 <ol><li> 자궁 경부 전위 또는 잘린 동물을 제물로 바친다. 하우스와는 동물 보호에 대한 제도 및 정부 정책에 따라 동물을 안락사. </li><li> 신속하게 일을 제거냉장 해부 블록에 두개골과 장소에서 전자 두뇌입니다. </li><li> 신선한 조직에서 해당 뇌 영역을 해부하고 3 단계로 진행합니다. </li></ol> 모터 구동 유리 테플론 균질와 3 조직의 균질화 <ol><li> 0.32 M HEPES 버퍼 자당 용액 4ml를 포함하는 레이블 13 ㎖의 폴리 프로필렌 튜브에 해부 조직 (SPM 용) 50 ~ 100 ㎎, 또는 (PSD 용) 100 ~ 200 mg의 놓습니다. 15 ㎖의 테이퍼 유리 - 테플론 조직 그라인더 / 균질화에 샘플을 이동시켜, 900 RPM (7 설정) 모터 드라이브를 설정하고 30 초에 걸쳐 12 스트로크 샘플을 균질화. 샘플 사이에 다른 유리 테플론 균질화를 사용하거나 샘플 사이의 차가운 증류수로 균질화를 씻어 킴으로 물기를 닦아냅니다. NOTE : 조직 4g 최대 그러나 그것은 적절한 분획을 확인하기 위해 이하의 조직을 사용하는 것이 바람직하다, 0.32 M 수크로오스 HEPES 완충 용액 4 ㎖에 균질화 될 수있다. </li><li> t 다시 균질화 샘플을 전송같은 13 ㎖의 폴리 프로필렌 튜브 오. 이사회 이후의 단백질 정량 및 총 단백질 분획의 서양 얼룩 분석을 위해 -80 ° C에서 파​​쇄 및 저장 100 ㎕. </li></ol> 4 저속 원심 분리가 핵 분획을 제거하는 (금리 뜨는 S1) <ol><li> 원심 분리기 4 ° C에서 10 분 동안 900 XG에 고정 각 회 전자의 균질. 새에 뜨는 (S1)을 전송, 13 ML 튜브를 표시하고 0.32 M HEPES 버퍼 자당의 500 μL에 핵 분수 펠렛 (P1)을 재현 탁. NOTE : (P1) 분획은 -80 ° C에 보관과 핵 분획의 웨스턴 블롯 분석을 위해 사용될 수있다. </li></ol> 원유 Synaptosomal 분수의 5 농축 (수익률 펠렛 P2) <ol><li> 4 ° C에서 15 분 동안 10,000 XG에 뜨는 (S1)을 원심 분리기. microcentrifuge 관에 500 ㎕의 후속 단백질 정량을위한 -80 ° C에서 저장하는 상층 액 (S2)와 예약을 제거동북 아시아의 평화와 번영과 세포질 / 라이트 막 분획의 서양 얼룩 분석. 0.32 M HEPES 버퍼 자당 용액 1 ㎖에 남아있는 원유 synaptosomal 분율 펠렛 (P2)을 재현 탁하고 또 다른 3 ㎖의 0.32의 M HEPES 버퍼 자당 솔루션을 추가합니다. </li><li> 4 ° C에서 15 분 동안 10,000 × g으로 원심 분리기. 이후 단백질 정량 및 세포질 / 라이트 막 분획의 서양 얼룩 분석을 위해 -80 ° C에서 저장하는 microcentrifuge 관에 뜨는 (S2 &#39;) 및 예비 500 μl를 제거합니다. 폴리 프로필렌 튜브에 세척 조 synaptosomal 펠렛 (P2 &#39;)을 저장합니다. </li></ol> 원유 Synaptosomal 분수의 6 용균 (Hypoosmotic 충격) <ol><li> DDH 2 O. 1 ㎖에 재현 탁을하여 폴리 프로필렌 튜브를 Lyse 조질 synaptosomal 펠렛 (P2 &#39;) DDH 2 O의 또 다른 3 ㎖를, 3 선을 손으로 유리 테플론 조직 균질로 전송하고, 균질화 추가합니다. 참고 : 그것은 중요빨리 가능한 빨리이 단계를 수행하고 6.2 단계로 진행합니다. </li><li> 다시 동일한 13 ml의 폴리 프로필렌 튜브에 빠르게 균질로부터 전송 샘플을, 1 M HEPES 용액 16 μL로 다시 4 mM의 HEPES으로 샘플을 조정 혼합 반전. </li><li> 30 분 완료 용균을 보장하기 위해 4 ° C에서 샘플을 돌립니다. </li></ol> Synaptosomal 막 분수의 7 농축 (P3) <ol><li> 4 ° C에서 20 분 동안 25,000 × g으로 원심 분리기. 조 기공 분수 상층 액 (S3)를 제거하고 이후의 단백질 정량 및 서양 얼룩 분석을 위해 -80 ° C에서 저장하는 5 ML 튜브에를 보유합니다. 0.32 M 수크로오스 HEPES 완충 용액 1 ㎖에 synaptosomal 세포막 분획 (P3)에 재현 탁. </li></ol> 불연속 자당 그라데이션의 8 준비 <ol><li> 피펫 정확히 3.5 ml의 1.2 M HEPES 버퍼 자당 용액 정확히 3.0 ㎖의 각 1.0 M 0.8 M HEPE의별도의 6 ML에 S-버퍼 자당 솔루션은 캡 튜브 웁니다. 참고 :이이 자당 기울기를 확인하는 데 사용되는 버퍼의 양을 미리 측정하기 위해 수행됩니다. 볼륨의 정확한 측정 결과 그라데이션 초 원심 분리에 대한 균형 될 수 있도록하는 것이 중요합니다. </li><li> 유리 파스퇴르 피펫 및 전구를 사용하여 12 ㎖의 polyallomer 초 원심 분리기 튜브에 1.2 M HEPES 완충 수크로오스 용액을 전송. 조심스럽게 1.2 M HEPES 버퍼 자당 솔루션의 상단에 1.0 M HEPES 버퍼 자당 솔루션을 층. 마지막으로, 1.0 M HEPES 버퍼 자당 솔루션의 상단에 0.8 M HEPES 버퍼 자당 솔루션을 층. 각 자당 솔루션에 대한 신선한 파스퇴르 피펫을 사용하고 자당 기울기를 방해하지 않도록주의. 벤치 소용돌이 또는 마이크로 원심에서 진동은 그라데이션의 무결성을 방해 할 것이다. </li><li> 파스퇴르 피펫을 이용하여 제조 된 불연속 자당 구배 상단에 재현 탁 synaptosomal 세포막 분획 (P3)를 층.그라디언트가 스윙 버킷 로터의 버킷 내부를 무게로 균형되어 있는지 확인합니다. 버킷 균형을하지 않은 경우, 조심스럽게 로터 버킷 그라데이션의 상단에 0.32 M HEPES 버퍼 자당을 추가하고 균형 P200 피펫을 사용합니다. </li></ol> 시냅틱 플라즈마 멤브레인의 9 분별 (SPM) <ol><li> 4 ° C에서 2 시간 동안 150,000 XG에 스윙 버킷 로터에서 초 원심 분리기. 조심스럽게 초 원심 분리기의 버킷에서 자당 구배를 제거합니다. 18 G 바늘과 1 ㎖의 주사기를 사용하여, 1.0 M / 1.2 M HEPES 완충 자당 용액의 계면의 하단에 튜브를 천공하고 시냅스 세포막 층 (SPM)을 철회. </li><li> 각 SPM 샘플 1 ML의 주사기에서 차지하는 부피를 기록합니다. 각 샘플이 조정되면 정확히 4 mM의 HEPES의 2.5 볼륨이 0.32 M.에 1.2 M의 자당 농도를 조정 추가, 3.5 ml의 두꺼운 벽 초 원심 분리기 튜브에 수집 된 SPM 층을 배치에드는 0.32 M 자당에, 0.32 M HEPES 버퍼 자당 용액으로 튜브를 균형. </li><li> 4 ° C에서 30 분 동안 200,000 XG에 고정 각 회 전자의 초 원심 분리기. 제거하고 상층 액을 버린다. 50 mM의 HEPES / 2 mM의 EDTA 용액 300 μL에 시냅스 세포막 펠릿 (SPM)을 재현 탁. -80 ° C에서 보관 샘플 서부 블로 팅에 사용되는 때까지. </li></ol> 시냅스 밀도 분획 (10) 제조 (PSD) <ol><li> 얼음에 SPM 샘플을 해동. </li><li> 50 mM의 HEPES / 2 mM의 EDTA 용액 0.54 % 트리톤 X-100 용액을 확인. </li><li> 5 ㎖의 폴리스티렌 관에서 트리톤 X-100 / HEPES / EDTA 용액 300 ㎕의 SPM 분율 2.7 ML을 결합하고 4 ° C에서 15 분 동안 샘플을 회전 </li><li> 4 ° C에서 20 분 동안 32,000 XG에 3.5 ㎖의 두꺼운 벽 초 원심 분리기 튜브와 원심 분리기 샘플로 전송 샘플. </li><li> -80에서 뜨는 (트리톤 X-100, 가용 분 [TS]) 및 저장을 예약; C를 °. 주 :이 단계에서는, 펠릿 &quot;PSD-1T&quot;분획을 나타낸다. 세제 초 처리는 &quot;PSD-2T&quot;부분을 생성합니다. PSD-1T 분율을 원하는 경우, 10.6 단계로 진행합니다. <ol><li> PSD-2T 분획을 생산하기 위해, 50 mM의 HEPES / 2 mM의 EDTA 용액 3 ㎖의 0.5 %의 트리톤 X에 펠릿을 재현 탁-100. 4 ° C에서 15 분 동안 샘플을 돌립니다. 4 ° C에서 20 분 동안 32,000 XG에 원심 분리기 샘플 및 10.6 단계로 진행합니다. </li></ol></li><li> 뜨는을 취소하고 50 mM의 HEPES / 2 mM의 EDTA 용액에 시냅스 (PSD) 펠렛을 재현 탁. NOTE : 재 부유 볼륨 펠릿의 크기에 의존 할 수있다; 50 ~ 75 μL의 볼륨을 권장합니다. PSD 분획 세제 불용 분획이기 때문에, 단백질의 완전한 재 부유 있도록 0.5 % SDS 1-2 μL를 추가하는 것이 필요하다. -80 ° C에서 보관 샘플 서부 블로 팅에 사용되는 때까지. </li></ol>

<ol>
	<li>Delint-Ramirez, I., 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=In+vivo+composition+of+NMDA+receptor+signaling+complexes+differs+between+membrane+subdomains+and+is+modulated+by+PSD-95+and+PSD-93.">In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 30, 8162-8170 (2010).</li><li>Wang, Q., 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=The+psychiatric+disease+risk+factors+DISC1+and+TNIK+interact+to+regulate+synapse+composition+and+function.">The psychiatric disease risk factors DISC1 and TNIK interact to regulate synapse composition and function.</a> Mol Psychiatry. 16, (2010).</li><li>Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinsertion+or+degradation+of+AMPA+receptors+determined+by+activity-dependent+endocytic+sorting.">Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting.</a> Neuron. 28, 511-525 (2000).</li><li>Tomita, S., Fukata, M., Nicoll, R. A., Bredt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+interaction+of+stargazin-like+TARPs+with+cycling+AMPA+receptors+at+synapses.">Dynamic interaction of stargazin-like TARPs with cycling AMPA receptors at synapses.</a> Science. 303, 1508-1511 (2004).</li><li>Ehlers, M. D., Heine, M., Groc, L., Lee, M. C., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusional+trapping+of+GluR1+AMPA+receptors+by+input-specific+synaptic+activity.">Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity.</a> Neuron. 54, 447-460 (2007).</li><li>Bats, C., Groc, L., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+Stargazin+and+PSD-95+regulates+AMPA+receptor+surface+trafficking.">The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking.</a> Neuron. 53, 719-734 (2007).</li><li>Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+level+controls+postsynaptic+composition+and+signaling+via+the+ubiquitin-proteasome+system.">Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system.</a> Nat Neurosci. 6, 231-242 (2003).</li><li>Cartier, E. 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=A+biochemical+and+functional+protein+complex+involving+dopamine+synthesis+and+transport+into+synaptic+vesicles.">A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles.</a> J Biol Chem. 285, 1957-1966 (2010).</li><li>Rodriguez de Lores, A., Alberici, M., De Robertis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+and+enzymic+studies+of+cholinergic+and+non-cholinergic+synaptic+membranes+isolated+from+brain+cortex.">Ultrastructural and enzymic studies of cholinergic and non-cholinergic synaptic membranes isolated from brain cortex.</a> Journal of Neurochemistry. 14, 215-225 (1967).</li><li>Gray, E. G., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+nerve+endings+from+brain:+an+electron-microscopic+study+of+cell+fragments+derived+by+homogenization+and+centrifugation.">The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation.</a> J Anat. 96, 79-88 (1962).</li><li>Cotman, C. W., Taylor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structural+studies+on+synaptic+complexes+from+rat+brain.">Isolation and structural studies on synaptic complexes from rat brain.</a> J Cell Biol. 55, 696-711 (1972).</li><li>Cotman, C. W., Banker, G., Churchill, L., Taylor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+postsynaptic+densities+from+rat+brain.">Isolation of postsynaptic densities from rat brain.</a> The Journal of Cell Biology. 63, 441-455 (1974).</li><li>Carlin, R. K., Grab, D. J., Cohen, R. S., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+postsynaptic+densities+from+various+brain+regions:+enrichment+of+different+types+of+postsynaptic+densities.">Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.</a> The Journal of Cell Biology. 86, 831-845 (1980).</li><li>Jones, D. H., Matus, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+synaptic+plasma+membrane+from+brain+by+combined+flotation-sedimentation+density+gradient+centrifugation.">Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation.</a> Biochim Biophys Acta. 356, 276-287 (1974).</li><li>Kennedy, M. B., Bennett, M. K., Erondu, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+immunochemical+evidence+that+the+"major+postsynaptic+density+protein"+is+a+subunit+of+a+calmodulin-dependent+protein+kinase.">Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 80, 7357-7361 (1983).</li><li>Moon, I. S., Apperson, M. L., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+tyrosine-phosphorylated+protein+in+the+postsynaptic+density+fraction+is+N-methyl-D-aspartate+receptor+subunit+2B.">The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 3954-3958 (1994).</li><li>Cho, K. O., Hunt, C. A., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+brain+postsynaptic+density+fraction+contains+a+homolog+of+the+Drosophila+discs-large+tumor+suppressor+protein.">The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.</a> Neuron. 9, 929-942 (1992).</li><li>Salahpour, 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=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc Natl Acad Sci U S A. 105, 4405-4410 (2008).</li><li>Ma, D. K., 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=Neuronal+activity-induced+Gadd45b+promotes+epigenetic+DNA+demethylation+and+adult+neurogenesis.">Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis.</a> Science. 323, 1074-1077 (2009).</li><li>Budreck, E. C., Scheiffele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin-3+is+a+neuronal+adhesion+protein+at+GABAergic+and+glutamatergic+synapses.">Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses.</a> The European Journal of Neuroscience. 26, 1738-1748 (2007).</li><li>Wu, J., 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=Arc/Arg3.1+regulates+an+endosomal+pathway+essential+for+activity-dependent+beta-amyloid+generation.">Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent beta-amyloid generation.</a> Cell. 147, 615-628 (2011).</li><li>Huttner, W. B., Schiebler, W., Greengard, P., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsin+I+(protein+I),+a+nerve+terminal-specific+phosphoprotein.+III.+Its+association+with+synaptic+vesicles+studied+in+a+highly+purified+synaptic+vesicle+preparation.">Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation.</a> J Cell Biol. 96, 1374-1388 (1983).</li><li>Nagy, A., Baker, R. R., Morris, S. J., Whittaker, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preparation+and+characterization+of+synaptic+vesicles+of+high+purity.">The preparation and characterization of synaptic vesicles of high purity.</a> Brain Res. 109, 285-309 (1976).</li><li>Cheng, D., 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=Relative+and+absolute+quantification+of+postsynaptic+density+proteome+isolated+from+rat+forebrain+and+cerebellum.">Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.</a> Molecular &amp; Cellular Proteomics : MCP. 5, 1158-1170 (2006).</li><li>Sheng, M., Hoogenraad, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+postsynaptic+architecture+of+excitatory+synapses:+a+more+quantitative+view.">The postsynaptic architecture of excitatory synapses: a more quantitative view.</a> Annu Rev Biochem. 76, 823-847 (2007).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a Laboratory Manual. , (1989).</li><li>Eggena, M., 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=Identification+of+histone+H1+as+a+cognate+antigen+of+the+ulcerative+colitis-associated+marker+antibody+pANCA.">Identification of histone H1 as a cognate antigen of the ulcerative colitis-associated marker antibody pANCA.</a> J Autoimmun. 14, 83-97 (2000).</li><li>Salahpour, 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=Homodimerization+of+the+beta2-adrenergic+receptor+as+a+prerequisite+for+cell+surface+targeting.">Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting.</a> J Biol Chem. 279, 33390-33397 (2004).</li><li>Bernocco, S., 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=Sequential+detergent+fractionation+of+primary+neurons+for+proteomics+studies.">Sequential detergent fractionation of primary neurons for proteomics studies.</a> Proteomics. 8, 930-938 (2008).</li><li>Grunewald, T. G., 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+localization+and+cytosolic+overexpression+of+LASP-1+correlates+with+tumor+size+and+nodal-positivity+of+human+breast+carcinoma.">Nuclear localization and cytosolic overexpression of LASP-1 correlates with tumor size and nodal-positivity of human breast carcinoma.</a> BMC Cancer. 7, 198 (2007).</li><li>Heimann, K., Percival, J. M., Weinberger, R., Gunning, P., Stow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+isoforms+of+actin-binding+proteins+on+distinct+populations+of+Golgi-derived+vesicles.">Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles.</a> J Biol Chem. 274, 10743-10750 (1999).</li><li>Neff, R. A., Gomez-Varela, D., Fernandes, C. C., Berg, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+scaffolds+for+nicotinic+receptors+on+neurons.">Postsynaptic scaffolds for nicotinic receptors on neurons.</a> Acta Pharmacol Sin. 30, 694-701 (2009).</li><li>Cella, N., Cornejo-Uribe, R. R., Montes, G. S., Hynes, N. E., Chammas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lysosomal-associated+membrane+protein+LAMP-1+is+a+novel+differentiation+marker+for+HC11+mouse+mammary+epithelial+cells.">The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells.</a> Differentiation. 61, 113-120 (1996).</li><li>Otera, H., 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=Peroxisomal+targeting+signal+receptor+Pex5p+interacts+with+cargoes+and+import+machinery+components+in+a+spatiotemporally+differentiated+manner:+conserved+Pex5p+WXXXF/Y+motifs+are+critical+for+matrix+protein+import.">Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import.</a> Mol Cell Biol. 22, 1639-1655 (2002).</li><li>Goubaeva, F., 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=Cardiac+mitochondrial+connexin+43+regulates+apoptosis.">Cardiac mitochondrial connexin 43 regulates apoptosis.</a> Biochem Biophys Res Commun. 352, 97-103 (2007).</li><li>Lamers, K. J., 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=Protein+S-100B,+neuron-specific+enolase+(NSE),+myelin+basic+protein+(MBP)+and+glial+fibrillary+acidic+protein+(GFAP)+in+cerebrospinal+fluid+(CSF)+and+blood+of+neurological+patients.">Protein S-100B, neuron-specific enolase (NSE), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) and blood of neurological patients.</a> Brain Res Bull. 61, 261-264 (2003).</li><li>Arunachalam, L., 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=Munc18-1+is+critical+for+plasma+membrane+localization+of+syntaxin1+but+not+of+SNAP-25+in+PC12+cells.">Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells.</a> Mol Biol Cell. 19, 722-734 (2008).</li><li>Brandstatter, J. H., Fletcher, E. L., Garner, C. C., Gundelfinger, E. D., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+presynaptic+cytomatrix+protein+bassoon+among+ribbon+synapses+in+the+mammalian+retina.">Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina.</a> Eur J Neurosci. 11, 3683-3693 (1999).</li></ol>

The authors have no conflicts of interest to disclose.

성공적인 결과를위한 중요한 절차의 몇 가지 단계가 있습니다. 단계 3에서는, 균질화의 일관성 정도가 각각의 샘플에 대해 달성되는 것이 중요하다. 구동 모터와 균질 조직을 균질화 할 때 일정한 속도 유봉의 회전뿐만 아니라, 스트로크의 수뿐만 아니라 사용된다. 저장성 용액 확장 균질화 또는 배양이 미토콘드리아를 용해하고 SPM 샘플을 오염 때문에 삼투압 충격 동안 배양 시간은 정확해야한?...

뉴런은 시냅스를 통해 통신하고,이 통신 품질이 시냅스에서 단백질 조성물의 변화가 큰 범위로 조절된다. 특히, 시냅스 후 밀도에있는 단백질은 신호 전달 체계 1 속속들이 비계 신경 전달 물질 수용체의 연결에 의해 통신에 참여한다. 또한 시냅스 효능의 강도 변화는 지속 시냅스 후 밀도 1-6에서 추가 또는 수용체의 제거에 의해 제어된다. 따라서 시냅스 단백질의 분리 및 정량은 신경 자극과 시냅스 효능 7을 변경하는 대응 방법에 대한 통찰력을 얻을 수있는 필요하고 유용한 기술이다. 이 문서에서는 불연속 자당 그라디언트에 초 원심 분리에 의해 쥐 뇌 조직에서 시냅스 단백질을 분리하는 일반적인 방법에 대해 설명합니다. 시냅스 세포막 분획을 농축 할 수 있고 기반 절연경험적 1.2 M 수크로오스 유사한 것으로 판정 된 수크로오스의 밀도. 생물학적 질문에 따라 세포 내 분획 자당 또는 퍼콜 하나의 연속 또는 불연속 그라디언트에 의해 분리 될 수있다. 연속 그라디언트 여러 분획으로 단백질의 분리를 허용; 이 주어진 분수 8 내에서 단백질의 공동 현지화을 설명하기 위해 특히 유용 할 수 있습니다. 그러나, 연속 그라디언트의 준비는 더 힘든이며 많은 응용 프로그램에 필요하지 않습니다. 불연속 구배가 비교적 쉽게 제조 할 수 있으며, 몇 일반적으로 정의 된 단백질 분획을 분리하는 데 사용될 수있다. 증가 몰 농도의 수크로오스 세 층으로 구성되는 불연속 구배 널리 시냅스 원형질막 (SPM)과 연관된 단백질을 분리하는데 사용되어왔다. 이 시냅스 세포막 분획을 상기 시냅스 후 밀도 fractio로 처리 될 수있다세제 불용성 부분의 세제 처리 및 분리에 의해 N (PSD). 이 프로세스가 첫번째 1960 9,10에서 설명 될 때, 전자 현미경은 대략 시냅스 세포막과 시냅스 후 밀도 분획 9-14을 정의하는 소기관 및 세포막을 입증하기 위해 사용되었다. 이러한 연구들은 전후 시냅스 막 및 SPM 분획 시냅스 소포의 포함을 증명; 세제 치료를 주로 전자 조밀 한 후, 시냅스 밀도를 볼 수 있었다. 절차에서, 저장성 충격 전지 본체 (10)로부터 시냅스 핀치 오프 공정을 위해 사용된다. 이 단계는 미토콘드리아는 삼투압 충격에 더 저항성을 그대로 유지하고, 그래서 그들은 자당 구배 (도 1)의 바닥에 침강한다는 사실을 이용한다. 이 같은 농축 기술을 사용하여, SPM 및 PSD 분수 생화학 첫번째이었다폴리 아크릴 아미드 겔 전기 영동 및 주요 단백질 성분 15-17의 서열에 의해 정의. 그 후 서양 얼룩 분석 (그림 2)이 분수를 정의 더 감지하고 시냅스 단백질의 수준을 정량화하는 데 사용되었습니다. 우리는 Slc6a3 궤적 쥐 18에서 중복 될 때 발생하는 도파민 수송의 시냅스 수준의 변화를 정량화하기 위해 실험실에서이 기술을 사용하고 있습니다. 우리는 또한 DISC1의 interactome 19의 일부 단백질이 시냅스 별 감축을 발견하는 NMDA 수용체 결핍 생쥐에서이 기술을 사용하고 있습니다. 또한 SPM 획분 시냅스 소포 막 단백질, 엔도 좀 마커, 미토콘드리아 단백질, 막 관련된 합성 효소 및 신호 전달 분자뿐만 아니라, 시냅스 후 밀도의 적분 요소 및 시냅스 세포막 (20-23)를 포함하는 웨스턴 블롯 분석으로부터 명백하다. E하지마 PSD 풍부한 분획 미토콘드리아 단백질 오염을 가질 수 있으며이를 제거하기 위해 13 초 구배 침강 또는 추가적인 정제 단계를 수행 할 필요가있다. 최근 정량적 질량 분석은 단독 시냅스 후 밀도의 100 위에 단백질의리스트뿐만 아니라, 이들 구성 요소 (24, 25)의 상대적인 풍요의 표시를 제공하고있다.

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			<td height="21" style="height:21px;width:261px;">1M HEPES, pH 7.4</td>
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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Glass Teflon homogenizer Kontes Duall 23</td>
			<td style="width:129px;">VWR</td>
			<td style="width:111px;">KT885450-0023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">IKA Model RW 16 Basic Stirrer</td>
			<td style="width:129px;">IKA Works</td>
			<td style="width:111px;">2572100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall SM-24 Fixed Angle Rotor</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">29017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall RC 6 Plus Centrifuge</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">46910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thinwall Polyallomer Tubes (13.2mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SW 41 Ti Rotor Swinging Bucket</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Beckman L-80 Floor Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thickwall Polycarbonate Tubes (3.5mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TLA-100.3 Fixed Angle Rotor</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349481</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:261px;">Beckman TL-100 Tabletop Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

신경 세포 내 분획 기술과 시냅스에서 인신 매매 단백질의 정량화 할 수 있습니다. 원래는 1960 년대 후반에 설명한 바와 같이, 시냅스 세포막과 연관된 단백질 자당 밀도 구배 초 원심 분리에 의해 단리 될 수있다. 시냅스 막 단리되면 시냅스 후 밀도로 알려진 고분자 복합체이어서 인해 세제 불용성으로 분리 될 수있다. 시냅스 세포막과 시냅스 후 밀도 단백질을 분리하는 데 사용되는 기술들은 본질적으로 사십년 후 동일하게 유지하고, 현재 널리 신경 과학 연구에 사용된다. 이 문서에서는 시냅스 세포막과 시냅스 밀도가 불연속 자당 기울기 사용과 관련된 단백질의 분별에 대해 자세히 설명합니다. 그 결과 단백질 제제는 서쪽 블로 팅 또는 2D DIGE 분석에 적합하다.

자당 밀도 구배의 제제는 수 크로스 (0.8, 1.0 및 1.2 M 수크로오스)의 세 몰 솔루션이 완전히 분리 될 것이다. 단백질 샘플이 추가되기 전에 그라데이션의 예는도 3a를 참조하십시오. 구배 미리 너무 제조하거나, 다른 기기로부터 진동 벤치 표면에 제조 된 경우, 구배는 손상 될 것이며, 적절한 분리가 달성 될 수없는 경우. 세 용액의 명확한 분리가 보이지 않는 경우, 단백질 시료를 추가?...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

이 문서에서는 불연속 자당 기울기에 초 원심 분리에 의해 시냅스 세포막과 관련된 단백질의 농축을 자세히 설명합니다. 시냅스 밀도 단백질의 준비 과정도 설명한다. 단백질 제제는 서쪽 블로 팅 또는 2D DIGE 분석에 적합하다.

불연속 자당 구배를 사용 시냅틱 세포막과 시냅스 밀도 단백질의 제조

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Research

JoVE Journal

Neuroscience

31.0K 조회수.  University of Toronto.  이 문서에서는 불연속 자당 기울기에 초 원심 분리에 의해 시냅스 세포막과 관련된 단백질의 농축을 자세히 설명합니다. 시냅스 밀도 단백질의 준비 과정도 설명한다. 단백질 제제는 서쪽 블로 팅 또는 2D DIGE 분석에 적합하다. 

비디오: Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Styryl 염료의 Photoconversion를 사용하여 시냅틱 소포 풀 유학

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

연구

신경과학

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

멤브레인 후보, 시냅틱 응답,의 연결 회로, Neuromodulation과 크레이를 사용하여 근육 조직학 : 학생 실험실 연습

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

불연속 Percoll - 자당 밀도 구배를 사용하여 마우스를 접속에서 Synaptoneurosomes의 준비

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

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 ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

성상 세포 운송업자 전류의 디컨 볼 루션 분석 글루타민산 정리의 시간 과정을 도출

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

신경 근육 접합 : 측정 시냅스 크기, 공 초점 형광 현미경을 사용하여 시냅스 단백질 농도의 분열 및 변경

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.