이 작품은 HEM에 NIH 교부금 DA15169 및 DA035224에 의해 투자되었다

모든 동물 취급 및 조직 수확은 승인 된 프로토콜 번호를 A1506 (멜리 키앙, PI) 다음, 매사 추세 츠 의과 대학 기관 동물 관리 사용위원회의 대학 (IACUC)의 지침에 따라 수행 하였다. 필수 솔루션 인공 뇌척수 (ACSF) - 신선한 매일 확인 125 밀리미터의 NaCl, 2.5 밀리미터의 KCl, 1.2 ㎜의 NaH 2 PO 4, 1.2 mM의 MgCl2를 2.4 mM의 염화칼슘, 26 mM의 탄산 수소 나트륨, 11 mM의 포도당 참고 : 배 원액으로 ACSF을 준비, 탄산 수소 나트륨과 포도당을 제외하고. 신선한 탄산 수소 나트륨과 포도당으로 보충, 배 주식에서 매일 1X 작업 솔루션을합니다. 자당이 첨가 ACSF (SACSF) - 신선한 매일 확인 250 ㎜의 SUC2.5 밀리미터의 KCl, 1.2 ㎜의 NaH 2 PO 4, 1.2 mM의 MgCl2를 2.4 mM의 염화칼슘, 26 mM의 탄산 수소 나트륨, 11 mM의 포도당을 증가 참고 : 배 원액으로 SACSF을 준비, 탄산 수소 나트륨과 포도당을 제외하고. 신선한 탄산 수소 나트륨과 포도당으로 보충, 배 주식에서 매일 1X 작업 솔루션을합니다. 설포-N-hydroxysuccinyl-SS - 비오틴 (설포-NHS-SS-비오틴, 피어스 케미칼) 주식 솔루션은 DMSO 200 ㎎ / ㎖하고 여러 동결 / 해동 사이클에 저항해야한다. 분액 -20 ° C.에 저장됩니다 숙시 닐 에스터 급속 수용액에서 가수 분해되므로, 작업 해법 전에 슬라이스에 적용 즉시 준비되어야한다. 슬라이스 담금질 솔루션 ACSF 100 mM의 글리신과 보충 RIPA 용해 BuffeR 10 mM 트리스, pH 7.4의 150 mM의 염화나트륨, 1.0 mM의 EDTA, 1 % 트리톤-X-100, 0.1 % SDS, 1 % 나 콜산 프로테아제 억제제와 RIPA (RIPA / PI) - 신선한 매일 확인 RIPA는 1 μM의 류 펩틴, 1 μm의 펩 스타틴, 1 μM의 아프로 티닌, 1 밀리미터 페닐 메틸 설 포닐 플루오 라이드로 보충. 1. 뇌 조각을 준비한다 <ol><li> 신선한 1X의 SACSF 및 1X ACSF을 </li><li> 작은 비커에 얼음에 SACSF을 풀어. 이 갓 수확 된 마우스의 두뇌를 유지하는 데 사용됩니다. </li><li> 95 % / 5 % O 2 / CO 2, 얼음에 20 분 버블 링에 의해 산소와 ACSF 및 SACSF 포화. </li><li> P30-38 마우스는 최적의 조직의 생존을 위해 사용되어야한다. 자궁 경부 전위와 잘린 동물을 희생하고 빠르게 prechilled, 산소 SACSF에 머리를 제거합니다. </li><li> 진동 마이크로톰을 사용하여, 그 지역에서 300 μm의 뇌 부분을. </li><li> 원하는 경우, 조각은 더 특정 뇌 영역 또는 별도 오른쪽으로 각각 제어 및 실험 슬라이스로 사용할 왼쪽 반구에 풍부하게 복구 이전 해부 할 수 있습니다. </li><li> 화재 광택 파스퇴르 피펫을 사용하여 24 - 웰 플레이트에 설정 챔버 바닥 메쉬에 조각을 전송합니다. </li><li> 슬라이스가 계속, 부드러운 버블 링, 산소 ACSF에서, 40 분 동안 31 ° C를 복구 할 수 있습니다. </li></ol> 2. 약물 치료 (해당되는 경우) 및 슬라이스 바이오 티 닐화 <ol><li> 복구 후, 예비 가온 (37 ° C), 산소 ACSF의 버블 링에서 지속적으로 95 % / 5 % O 2 / CO 2 조각을 씻어 배. </li><li> 시험 물질을 추가하고 지속적으로 산소와 부화. <ol><li> 편의를 위해, 10 배 농축 된 약물의 1 / 10 번째 볼륨을 추가하고 부드럽게 반전 섞는다. </li><li> 온화하게 원하는 온도에서 수욕 플레이트를 흔들어. </li></ol></li><li> 다음 약물 치료, 급속차가운 얼음 ACSF에 배를 세척하여 냉기의 조각. </li></ol> 3. Biotinylate 표면 단백질 <ol><li> 바로 전에 라벨에 차가운 얼음 ACSF의 준비 1.0 ㎎ / ㎖ 설포-NHS-SS - 비오틴. </li><li> 슬라이스 설포-NHS-SS - 비오틴 825 mL를 넣고 얼음, 45 분에 슬라이스를 품어. </li><li> 얼음에 차가운 얼음 ACSF에서 10 분 동안 배양 한 다음, 얼음 차가운 ACSF 빠르게 조각을 세 번 씻으십시오. </li><li> 차가운 얼음 조각 끄다 버퍼 조각을 세 번 세척하고 무료 설포-NHS-SS - 비오틴을 종결 825 ㎖의 슬라이스 급냉 버퍼와 얼음에 두 번, 25 분을 품어. </li></ol> 4. 조직 해물을 준비 <ol><li> 차가운 얼음 ACSF의 조각을 세 번 세척하고 화재 광택 파스퇴르 피펫을 사용하여 microcentrifuge 관에 각 조각을 전송합니다. </li><li> 부드럽게 200 XG, 1 분 조심스럽게 대기음 나머지 ACSF을 원심 분리하여 슬라이스를 펠렛. </li><li> 400 μL 차가운 얼음 RIPA / PI를 추가로 pipetting하여 조직을 파괴하고 아래로 한 번 P200 핍을 통해ETTE. </li><li> 전송 신선한 튜브 조각 / RIPA를 분해 및 용해를 완료하기 위해, 회전, 30 분, 4 ° C를 품어. </li><li> 원심 분리하여 세포 파편 펠렛, 18,000 XG, 15 분, 4 ° C. </li><li> 표준으로 소 혈청 알부민 (BSA)와 BCA 단백질 분석법을 사용하여 용 해물의 단백질 농도를 결정한다. </li></ol> 5. 바이오틴 단백질을 분리 <ol><li> 비드 / 총 단백질 비율을 최적화합니다. 주 : 개별 단백질 발현 수준은 다른 뇌 영역에 걸쳐 광범위하게 변할 수있다. 조직 파쇄물의 주어진 양에서 바이오틴 단백질 모두가 캡처하지 않는 한, 정확하게 표면 발현의 잠재적 인 변화를 감지 할 수 없다. 따라서, 경험적 전에 새로운 슬라이스 바이오 티 닐화 연구 착수시에 특정 단백질 / 뇌 영역에 대한 최적의 비드 / 전체 단백질의 비율을 결정하기 위해 필수적이다. <ol><li> 증가 금액으로 25 ㎕의 스트렙 타비 딘 아가로 오스의 구슬을 품어, 세척 및 용출 단계를 바인딩 그런 다음 아래의 설명에 따라 조직 용 해물 (대략적인 범위 25 ~ 200 μg), 그리고의 진행. </li><li> 그 결과 면역 반응 대역을 정량화하고 그 증가 또는 단백질의 표면 발현을 감소 하나의 정확한 정량화를 허용합니다 바인딩의 선형 범위의 비드 / 해물 비율을 선택합니다. </li></ol></li><li> 스트렙 타비 딘 - 아가로 오스의 구슬을 준비 <ol><li> 모든 샘플에 필요한 총 비드 볼륨을 결정합니다. 25 μL / 샘플 = 100 μl의 구슬 + 하나 추가 = 125 μl의 구슬에 그 금액에 대한 충분한 비드 볼륨을 더한 추가 (즉, 4 샘플을 준비합니다. </li><li> 소용돌이 스​​트렙 타비 딘 구슬 주식 및 피펫 밖으로 볼륨을 원하는. P200 피펫을 사용하면, 방해 또는 구슬의 손상을 방지하기 위해 팁 단부를 잘라. </li><li> 객실 성질에서 18,000 XG, 1 분을 원심 분리하여 방부제, 각 RIPA 추가 후 소용돌이로 교반 및 세척 사이에 수집 구슬을 제거하기 위해 0.5 ~ 1.0 ㎖의 RIPA / PI에 구슬을 3 회 세척ature. </li><li> 진공 플라스크에 부착 된 유리 파스퇴르 피펫을 사용하여 세척 사이의 버퍼를 대기음. 흡입시 유리 파스퇴르 피펫의 단부에 부착 플라스틱 P200 팁은 미세한 제어를 제공한다. 이 피펫으로 흡입 비즈 위험으로는, 우선이 세척 절대적 모든 버퍼를 제거 할 필요가 없다. 최종 세척 후 구슬을 흡입하지 않고 가능한 한 많은 여분의 버퍼를 제거합니다. </li><li> 과에 resuspend 아래로 피펫 팅, 다시 원래 볼륨으로 구슬을 가지고 RIPA / PI를 추가합니다. 비즈 튜브 벽에 붙어있는 바와 같이,이 시점에서 소용돌이로 교반을 피하십시오. </li><li> 끝으로 P200을 사용하여 마이크로 원심 튜브로 나누어지는 비즈 차단. 구슬이 균등하게 중단과 튜브 사이에 분산 남아 보장하기 위해 다운 샘플링 사이에 몇 번을 피펫. </li></ol></li><li> 구슬을 스트렙 타비 딘에 바인딩 바이오틴 단백질 <ol><li> 아가로 오스 비즈가 들어있는 튜브에 세포 용 해물을 배포합니다. 실험에 갔지<html> 여러 샘플을 비교되고 다시 정확한 비교를 위해 각 샘플에 대해 동일한 양의 단백질을 사용해야합니다. </li><li> 200 ㎕의 최소한의 볼륨에 샘플을 가지고 추가 RIPA / PI를 추가합니다. 이 샘플을 배양하는 동안 적절하게 혼합하고 또한 샘플에 걸쳐 단백질 농도를 통합 할 확신합니다. </li><li> 관 회전 장치에 튜브를 넣고 4 ℃에서 하룻밤 혼합 </li><li> 별도의 튜브에 각각의 샘플에 사용되는 다운로드 용 해물 부피의 등가 분배. 샘플 볼륨이 높은 경우에 대안 적으로, 전체 용해질 부피의 분획을 SDS-PAGE 겔에 최대 부하 볼륨을 수용하기 위해, 대신에 할당 될 수있다. 이들은 총 단백질의 양을 표면 발현을 정상화하는 데 사용됩니다. </li><li> 같은 배의 볼륨이나 배의 SDS-PAGE 샘플 버퍼 1 / 5 볼륨과 하나의 샘플을 분석 할 때까지 -20 ° C에서 비드 샘플, 또는 매장과 동시에 4 ° C, 부화 하나를 추가합니다. </li></ol></li></ol>ove_title &quot;&gt; 6. 용출 및 샘플 분석 <ol><li> 실온에서 18,000 XG, 2 분 원심 분리하여 구슬 펠렛. </li><li> 기음 뜨는 세척 구슬 825 ㎖의 RIPA 3 회. 비드 손실을 최소화하기 위해, 세척 간 비즈 상기 버퍼의 작은 머리 부피를 남긴다. 최종 세척 후, 비드 펠렛을 방해하지 않고, 가능한 한 많은 RIPA를 제거합니다. </li><li> 이황화 결합을 감소시켜 스트렙 타비 딘 비즈에서 바이오틴 단백질을 용출. 샘플 버퍼, 소용돌이도를 감소 25 μL 2X SDS-PAGE를 추가하고 샘플 30 분, 실내 온도를 돌립니다. 참고 : 대부분의 막 단백질이 심각 그들의 전기 영동 이동성을 손상, SDS-PAGE 샘플 버퍼에 삶은 때 집계 높은 경향이있다. 이 조사되고 단백질 경우라면, 가열 샘플을 피하고, 대신에, 실온에서 서서히 용출. 비등 (병렬로 평가 다른 단백질이 비등을 필요로하는 경우 예) 절대적으로 필요한 경우, SAMPLE 버퍼는 응집을 최소화하기 위해 2 M 우레아 (최종 농도)로 보충 될 수있다. 어떤 경우에는 응집을 감소시키는 효과가 있지만, 요소는 종종 밴드의 모습을 타협. </li><li> 면역 블롯에 의해 샘플을 분석합니다. </li><li> 다운로드 용 해물 샘플을 해동하고 비드 샘플, 30 분간 실온에 평행하게 회전한다. </li><li> SDS-PAGE 젤에 별도의 단백질. </li><li> 면역 블로 그 단백질 (들)을 확인합니다. <ol><li> 밴드가 적절한 정량 검출의 선형 범위에서 검출되어 있는지 확인하십시오. </li><li> 바이오 티 닐화 시약이 손상 / 손상 세포를 통해 세포 내 단백질에 대한 액세스 권한을 얻게되지 않았 음을 보장하기 위해. 이것은 가장 조사되는 세포 유형에 특정한 세포 내 단백질을 위해 평행으로 면역 블에 의해 수행된다. </li><li> 밴드 밀도를 정량화 총 단백질 발현 수준의 비율로 상대 단백질 표면 밀도를 산출한다. </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+trafficking+and+the+plasticity+of+excitatory+synapses.">Receptor trafficking and the plasticity of excitatory synapses.</a> Curr. Opin. Neurobiol. 12, 279-286 (2002).</li><li>Bredt, D. S., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+at+excitatory+synapses.">AMPA receptor trafficking at excitatory synapses.</a> Neuron. 40, 361-379 (2003).</li><li>Kerchner, G. A., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silent+synapses+and+the+emergence+of+a+postsynaptic+mechanism+for+LTP.">Silent synapses and the emergence of a postsynaptic mechanism for LTP.</a> Nat. Rev. Neurosci. 9, 813-825 (2008).</li><li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+and+synaptic+plasticity.">AMPA receptor trafficking and synaptic plasticity.</a> Annu. Rev. Neurosci. 25, 103-126 (2002).</li><li>Borgland, S. L., Malenka, R. C., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+cocaine-induced+potentiation+of+synaptic+strength+in+the+ventral+tegmental+area:+electrophysiological+and+behavioral+correlates+in+individual+rats.">Acute and chronic cocaine-induced potentiation of synaptic strength in the ventral tegmental area: electrophysiological and behavioral correlates in individual rats.</a> J. Neurosci. 24, 7482-7490 (2004).</li><li>Dong, Y., 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=Cocaine-induced+potentiation+of+synaptic+strength+in+dopamine+neurons:+Behavioral+correlates+in+GluRA(-/-)+mice.">Cocaine-induced potentiation of synaptic strength in dopamine neurons: Behavioral correlates in GluRA(-/-) mice.</a> PNAS. 101, 14282-14287 (2004).</li><li>Hyman, S. E., Malenka, R. C., Nestler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+addiction:+the+role+of+reward-related+learning+and+memory.">Neural mechanisms of addiction: the role of reward-related learning and memory.</a> Annu. Rev. Neurosci. 29, 565-598 (2006).</li><li>Thomas, M. J., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+the+mesolimbic+dopamine+system.">Synaptic plasticity in the mesolimbic dopamine system.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358, 815-819 (2003).</li><li>Sorkina, T., Hoover, B. R., Zahniser, N. R., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+and+protein+kinase+C-induced+internalization+of+the+dopamine+transporter+is+mediated+by+a+clathrin-dependent+mechanism.">Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism.</a> Traffic. 6, 157-170 (2005).</li><li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical,+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat. Neurosci. 8, 881-888 (2005).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J. Biol. Chem. 278, 22168-22174 (2003).</li><li>Melikian, H. E., Buckley, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+trafficking+regulates+the+activity+of+the+human+dopamine+transporter.">Membrane trafficking regulates the activity of the human dopamine transporter.</a> J. Neurosci. 19, 7699-7710 (1999).</li><li>Daniels, G. M., Amara, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+trafficking+of+the+human+dopamine+transporter.+Clathrin-mediated+internalization+and+lysosomal+degradation+in+response+to+phorbol+esters.">Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters.</a> J. Biol. Chem. 274, 35794-35801 (1999).</li><li>Sorkina, 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=RNA+interference+screen+reveals+an+essential+role+of+Nedd4-2+in+dopamine+transporter+ubiquitination+and+endocytosis.">RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.</a> J. Neurosci. 26, 8195-8205 (2006).</li><li>Eriksen, 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=Visualization+of+dopamine+transporter+trafficking+in+live+neurons+by+use+of+fluorescent+cocaine+analogs.">Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs.</a> J. Neurosci. 29, 6794-6808 (2009).</li><li>Rao, A., Simmons, D., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+subcellular+distribution+of+endosomal+compartments+and+the+dopamine+transporter+in+dopaminergic+neurons.">Differential subcellular distribution of endosomal compartments and the dopamine transporter in dopaminergic neurons.</a> Mol. Cell Neurosci. 46, 148-158 (2011).</li><li>Zhao, 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=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nat. Methods. 8, 745-752 (2011).</li></ol>

저자는 재정적 이익에게이 작품과 충돌이 없습니다.

두뇌에 매우 영향 시냅스 신호 이입 매매, 그것은 양적으로 성인 신경 세포의 표면 단백질 발현의 변화를 측정하기 어려운 입증했다고 오랜 지식에도 불구하고. 이 작품에서 우리는 급성 뇌 조각의 표면 단백질 생체 레이블을 할 수있는 신뢰할 수있는 방법을보고합니다. 그들은 시냅스 연결과 자신의 준비 시간 이후에 세포 생존을 위로 유지로 뇌 조각의 준비는, 전기 생리학 레코딩을위한...

세포 내 이입 매매는 필수적인 막 단백질의 다양한 세포막의 프레 젠 테이션을 미세 조정 유비쿼터스 세포 메커니즘입니다. 엔도 시토 시스는 세포 내 환경 1에 필수 영양소를 제공하며, 수용체 활성화 2에 대한 응답으로 수용체 신호 둔감해진다. 확대 원형질막에 리사이클 이입은 별도로 세포 표면 (3)에 단백질 발현 수준을 증가시킴으로써 세포 신호 전달을 향상시킬 수있다. 또한, 멤브레인 인신 매매의 교란은 단백질의 세포 내 이입 매매를 제어하는 분자 메커니즘을 조사 할 필요성을 강조, 수많은 질병과 병적 인 조건 4,5에 연루되어있다. 많은 단백질이 지난 몇 년 동안 증거를 장착 고전 방 clathrin 의존 국제화 메커니즘을 사용하는 동안 여러 방 clathrin 독립적 인 세포 내 이입 메커니즘의 증가 배열의 세포 내 이입 가능성을 제어 것을 보여줍니다단백질 6,7. 따라서, 생리 관련 시스템에서 인신 매매를 촉진하는 세포 내 이입 메커니즘을 조사 할 필요가 상당히 성장했습니다. 뇌의 수용체, 이온 채널과 신경 전달 물질 수송의 세포 내 이입 매매는 시냅스 가소성 8-11 궁극적으로 신경 세포의 흥분성 시냅스 반응에 영향을 남용 12-15의 약물에 반응을 수립 기본 역할을하고 있습니다. 신경 세포의 인신 매매의 대부분의 연구는 이종 발현 시스템 또는 양식 기본 뉴런 하나에 의존하는 현재까지, 어느 것도 확실하게 성인 신경 세포 재생의 메커니즘을 반영 할 수있다. 여기, 우리는 양적으로 성인 설치류에서 파생 된 급성 뇌 조각의 표면 단백질의 수준을 측정하는 표면의 바이오 티 닐화를 사용하는 방법을보고합니다. 이 방법을 사용하여, 우리는 마우스 선조체 도파민 수송 체에 재 빠르게 내부화 입증 자료를 제시포르 볼 에스테르 - 매개 단백질 키나제 C (PKC)의 활성화에 sponse.

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

레귤 이입 매매 동적 분의 시간 스케일에서 수용체, 이온 채널 및 전달체 세포 표면 프레젠테이션을 제어함으로써, neuromodulatory 다양한 이벤트를 용이 중앙 메커니즘이다. 개별 단백질의 세포 내 이입 매매를 제어 메커니즘의 폭 넓은 다양성이있다. 인신 분자 토대를 조사 연구는 주로 정량적 외인성 자극 및 유전자 조작에 응답하여 세포막 단백질의 표면 발현의 변화를 측정하는 표면 바이오 티 닐화에 의존했다. 그러나,이 방법은 주로 충실히 성숙한 신경 세포 재생에 중요한 생리 학적 기전을 반영하지 않을 수도 배양 된 세포로 국한되었다. 또한, 배양 세포의 접근은 인신 매매 메커니즘의 지역별 차이를 과소 평가 할 수 있습니다. 여기, 우리는 급성 뇌 조각 준비에 세포 표면의 바이오 티 닐화를 확장하는 방법을 설명합니다. 우리이 방법은 성숙한 신경 세포에서 세포막 단백질 표면 수준의 급격한 변화를 측정하는 고 충실도 접근법을 제공한다는 것을 보여준다. 이 방법은 신경 세포 내 이입 매매의 분야에서 광범위한 유틸리티를 가질 가능성이있다.

신경원 도파민 수송 체는 세포 라인 16-20에서 PKC의 활성화에 응답 내재화된다. 세포주 및 발현 시스템의 다양한 PKC 유도 DAT 표면 손실을 보여주는 많은보고에도 불구하고, 배양 도파민 뉴런 21-23에서 이러한 발견을 확인하기 위하여 도전되었다. 우리는 직접 D​​AT 성인 도파민 신경 세포에서 PKC의 활성화에 대한 응답으로 내면화 여부를 테스트하기 위해 마우스 선조체 조각을 사?...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

신경 세포막 매매 동적 세포막 단백질 가능 크게 영향 신경 전달을 제어한다. 지금까지, 그것은 성인의 신경 세포에서 신경 세포 내 이입 매매를 측정하기 위해 도전하고 있습니다. 여기, 우리는 급성 뇌 조각의 표면 단백질 발현 생체의 급격한 변화를 측정 할 수있는 매우 효과적인, 양적 방법을 설명합니다.

뇌 슬라이스 바이오 티 닐화 :<em&gt; 전의 VIVO</em성인 뉴런에서 지역 고유의 플라즈마 막 단백질 인신 매매를 측정하는&gt; 접근

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

12.8K 조회수.  University of Massachusetts Medical School.  신경 세포막 매매 동적 세포막 단백질 가능 크게 영향 신경 전달을 제어한다. 지금까지, 그것은 성인의 신경 세포에서 신경 세포 내 이입 매매를 측정하기 위해 도전하고 있습니다. 여기, 우리는 급성 뇌 조각의 표면 단백질 발현 생체의 급격한 변화를 측정 할 수있는 매우 효과적인, 양적 방법을 설명합니다. 

비디오: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

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 ...

연구

신경과학

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

이미징은 기본 양식 마우스 뉴런의 플라즈마 막에 수용체 삽입을 pHluorin 태그

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

GABA 수용체 막 현지화 및 인신 매매를 연구하는 α-Bungarotoxin 바인딩 사이트 태그를 사용하여

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

슬라이스 뜨거운 것이 : 생리적 온도에서 급성 성인 뇌 슬라이스

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

뇌 조각의 공포 회로의 예 생체 Optogenetic 해부

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

마우스 기본 Hypothalamic 뉴런 Deconvolution 현미경을 사용 하 여 매매 GLUT4 단백질의 라이브 이미지

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Neurodegeneration Ex Vivo 쥐 두뇌 분할 영역을 사용 하 여 기본 이벤트를 공부 하는 양자 택일 접근

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

전 비보 내 분 비 신호 초파리 에 두뇌 응답을 시각화 칼슘 이미징

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

근 적외선 형광 및 고 분해능 주사를 사용 하 여 설치류 뇌의 단백질 발현 측정

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...