This work was supported by USPHS grants MH-091460 (E.N.), MH-084018 (A.S.), MH-094268 Silvo O. Conte center (A.S.), MH-069853 (A.S.), MH-085226 (A.S.), MH-088753 (A.S.), MH-092443 (A.S.), and MH-096208 (K.I.), grants from DANA (E.N.), Stanley (A.S.), RUSK (A.S.), S-R foundations (A.S.), NARSAD (A.S. and K.I.), and Maryland Stem Cell Research Fund (A.S. and K.I.).
We sincerely appreciate the efforts and contributions of Pearl Kim, Maria Papapavlou, Nao Gamo, Youjin Chung, Yukiko Lema and Mark Christie towards coordination of the biopsy process.

참고 :이 연구의 모든 연구 자원 봉사자는 하워드 대학의 심사위원회 (Institutional Review Board) 및 존스 홉킨스 대학의 승인을 동의 문서를 투여 하였다. 동의 문서에 서명함으로써 동의 만 참가자들은 연구에 등록되었다. 이 분석을위한 연구 기반을 구성 : 20 명 (12 BD 10 컨트롤, 30 % 남성)과 38.2 (14.1) 년의 나이 (SD)를 의미한다. 참고 : 유리 및 플라스틱 표면에서의 RNase 오염을 제거의 RNase 보내 겠 모든 표면을 스프레이. 1. 조직 검사 절차 <ol><li> 연구 참가자의 비강에 국소 리도카인 (xylocaine) 액체 4 %와 oxymetazoline 염산 0.05 % 스프레이와 시간을 마비의 15 분을 할 수 있습니다. </li><li> 리도카인과 oxymetazoline의 혼합물에 ½ × 3 인치 수술 cottonoids을 만끽하고 비중격의 우수한 부분을 따라 비강에 삽입하고 15 분 동안 앉아 할 수 있습니다. </li><li> 30 ° 강성을 사용하여비강 내시경, 비강의 상부를 찾아 여러 개의 작은 표본을 고려하여 비중격의 우수한 부분에서 최대 물고 코 집게를 사용하여 코 점막을 제거합니다. </li></ol> 조직 블록 2. 제조 <ol><li> 직전 생검 조직이 매체를 동결 표준 크기의 cryomolds를 입력합니다. </li><li> 멸균 핀셋을 사용하여, 동결 배지에서 신선한 조직을 탑재하고 드라이 아이스상에서 정지. , 냉동 매체 cryoblock의 나머지 부분을 채우기 드라이 아이스에 cryoblock을 동결하고, -80ºC에서 저장 블록. </li></ol> 3. 냉동 절편 <ol><li> 냉동 절편 직전 폴리에틸렌 나 프탈레이트 (PEN) 막 유리 슬라이드를 준비한다. 5 분의 RNase 보내 겠 솔루션으로 슬라이드를 스프레이. 그런 디 에틸 피로 카보 네트 (DEPC) 물에 슬라이드를 씻어 30 분간 자연 건조를 할 수 있습니다. 30 분 동안 UV 빛 아래 건조 슬라이드를 활성화합니다. </li><li> 동결 절단 (Cryosections)는 각각 30 μm의에서 완전히 차단합니다. 건조 i에서 슬라이드를 유지CE 및 섹션은 가능한 한 많은 냉동. 완료시 -80 ºC에서 보관 슬라이드. </li></ol> 4. 레이저 캡처 이외에 Microdissection <ol><li> 직전 미세 절제에 슬라이드를 탈수하여 시작합니다. 60 % (2 ㎖, 15 초), 80 % (2 ㎖, 15 초), 95 % (3 ㎖, 25 초), 100 : 각 슬라이드에 다음 에탄올 시리즈를 적용 한 후 냉동 슬라이드하자 10 초간 해동하고, % (7 ㎖를, 60 초). </li><li> 에 광 활성화 현지화 현미경 (PALM) 현미경을 켜고 설정 다음 조건에 : (82)에 에너지를 설정 10 배 배율의 경우, (74)에 초점을 맞추고 속도 25 - 30 20 배 배율의 경우, (63)에 에너지를 설정, 67에 초점 속도 및 5 - 6. 최적의 절단 결과를 얻기 위해 필요한만큼 조정한다. </li><li> 시각적으로 20 배 또는 10 배의 배율을 사용하여 신경 세포를 식별합니다. 염색 과정의 사용없이 현미경 기본 서브 점막 표면 후각 뉴런 얇은 층을 구별. 조밀 한 기둥 모양 (18)로 신경 세포를 관찰합니다.</li><li> 4.2 단계에서 상기 한 현미경에서 연필 기능을 사용하여 신경 층을 중심으로 추적. 레이저에서 신경 세포의 연소를 방지하기 위해 약간 떨어져 뉴런에서 추적합니다. 이는 고품질의 RNA를 얻기 위해 필수적이다. 그런 다음 레이저 유도 절단을 시작합니다. </li><li> 좋은 팁 미세 절제 집게를 사용하여 절개 섹션을 들고 얼음에 100 μL 용해 버퍼를 포함하는 마이크로 튜브에 조직을 넣어. 하나의 샘플에서 얻은 모든 신경 부분에 대해 반복합니다. 50 분을 초과 할 수 없습니다 샘플 당 총 해부 시간. </li><li> 해부가 완료 즉시 소용돌이 샘플 해물과 드라이 아이스 계속되면. 총 RNA 격리를위한 -80ºC에서 보관 샘플. </li></ol> 총 RNA 및 품질 관리 분석 5. 분리 <ol><li> 얼음에 해동 샘플 해물. 볼 (수정없이 업체로의 프로토콜에 따라 DNase의-I의 불 활성화와 RNAqueous 마이크로 키트 총 RNA 분리를 수행합니다 &lt;강한&gt; 그림 1). 용출 총 부피는 약 20 μL이다. </li><li> RNA의 농도를 측정하고 Bioanalyzer를 사용하여 RNA 무결성 번호 (RIN)와 RNA의 품질을 평가한다. 도 2의 품질 관리 기준을 만족 샘플 cDNA 합성에 사용될 수있다. </li></ol> 6. cDNA를 합성 <ol><li> 검증 된 상용 키트를 사용하여 cDNA를 합성 (시약의 표 참조). 튜브 당 8 ㎕의 총 부피로 RNA (이용 가능성에 따라)의 RNase없는 물과의 NG 1.0 - 0.1를 조합하여 어닐링 공정을 수행한다. 그런 다음 10 μL의 총 볼륨, 튜브 당 혼합 올리고 D (T) 20 프라이머 1 μL, 10 mM의 dNTP의 1 μl를 추가합니다. </li><li> 5 분 65 ° C에서 부드럽게 소용돌이와 원심 분리기 샘플 및 어닐링 샘플. 얼음에 반응이 종료 된 후, 즉시 냉각 샘플. </li><li> 표 1의 사양에 따라 마스터 믹스 솔루션을 준비합니다. 그런 다음 9.5 μ를 추가각 튜브에 L 마스터 믹스 및 상업적으로 제조 된 역전사 효소 (RT)의 0.5 μL. 대신 RT의 관을 제어하는​​ 0.5 μL의 RNA 분해 효소가없는 물을 추가합니다. </li><li> 없이 자전거로, 50 분, 5 분 및 최종 4 ºC 유지를위한 다음 85 ºC 50 ºC에서 처음 배양과 RT 반응을 실행합니다. </li><li> 물과 나누어 함께 5 배의 모든 샘플을 희석. -80 ºC에서 보관 샘플. </li></ol> 7. 리얼 타임 PCR - 신경 농축 확인 <ol><li> . 표 2의 사양에 따라 마스터 믹스 솔루션을 준비 같은 GAPDH와 같은 내부 통제를 사용합니다. 신경 농축을 결정하기 위해 undissected 조직에 OMP 식 microdissected 조직에서 후각 마커 단백질 (OMP) 식을 비교. 참고 : OMP는 후각 신경 세포 마커입니다. OMP 서열 포워드, 5&#39;- CTGTCGGACCTGGCCAAG-3 &#39; , 역 5&#39;-CACCCTCGCCAAAGGTGA-3 &#39; GAPDH 순서 : 앞으로, 5&#39;-CATGAGAAGTATGACAACAGCCT-3 &#39; 역, 5&#39;-AGTCCTTCCACGATACCAAAGT-3 &#39;. </li><li> cDNA를 각 웰 3 μL에 마스터 믹스의 7 μl를 추가하여 PCR 플레이트를 설정합니다. </li><li> 300 XG (1,300 RPM)에서 5 분 동안 원심 분리 판. 이러한 SYBR 푸르 qPCR의 상업적인 수퍼 믹스 지정된 기본 열 사이클링 조건을 이용하여 실행을 리얼 타임 PCR 반응. </li><li> 데이터를 (사양 그림 2 참조) 분석합니다. </li></ol> 8. 리얼 타임 PCR - 관심의 유전자의 발현 <ol><li> 2 배 이상의 신경 세포의 농축을 보여 샘플을 수행합니다. </li><li> 각 프로브 표 3의 사양에 따라 별도의 튜브 (이자 및 VIC 표지 GAPDH의 FAM 표지 프로브) 얼음에 마스터 믹스를 준비합니다. </li><li> cDNA를 각 웰과 2 μL 8 μL 마스터 믹스 PCR 플레이트를 설정합니다. </li><li> 300 XG (1,300 RPM)에서 5 분 동안 접시를 원심 분리기. DEF를 사용하여 실시간-PCR 반응을 실행제조자의 유전자 발현 마스터 믹스 프로토콜에 따라 AULT 열 사이클링 조건. 식의 결과를 (사양 그림 2 참조) 분석합니다. </li></ol>

<ol>
	<li>Goodwin, F. K., Jamison, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manic-depressive illness. , (1990).</li><li>Einat, H., Manji, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+plasticity+cascades:+genes-to-behavior+pathways+in+animal+models+of+bipolar+disorder.">Cellular plasticity cascades: genes-to-behavior pathways in animal models of bipolar disorder.</a> Biol Psychiatry. 59 (12), 1160-1171 (2006).</li><li>Severino, 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=Pharmacogenomics+of+bipolar+disorder.">Pharmacogenomics of bipolar disorder.</a> Pharmacogenomics. 14 (6), 655-674 (2013).</li><li>Beech, R. 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=Gene-expression+differences+in+peripheral+blood+between+lithium+responders+and+non-responders+in+the+Lithium+Treatment-Moderate+dose+Use+Study+(LiTMUS).">Gene-expression differences in peripheral blood between lithium responders and non-responders in the Lithium Treatment-Moderate dose Use Study (LiTMUS).</a> Pharmacogenomics J. 14 (2), 182-191 (2013).</li><li>Horiuchi, 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=Olfactory+cells+via+nasal+biopsy+reflect+the+developing+brain+in+gene+expression+profiles:+utility+and+limitation+of+the+surrogate+tissues+in+research+for+brain+disorders.">Olfactory cells via nasal biopsy reflect the developing brain in gene expression profiles: utility and limitation of the surrogate tissues in research for brain disorders.</a> Neurosci Res. 77 (4), 247-250 (2013).</li><li>Dryer, L., Graziadei, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+the+olfactory+bulb+in+an+elasmobranch+fish,+Sphyrna+tiburo:+segregation+of+inputs+in+the+telencephalon.">Projections of the olfactory bulb in an elasmobranch fish, Sphyrna tiburo: segregation of inputs in the telencephalon.</a> Anat Embryol (Berl. 190 (6), 563-572 (1994).</li><li>Hahn, C. 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=In+vivo+and+in+vitro+neurogenesis+in+human+olfactory+epithelium.">In vivo and in vitro neurogenesis in human olfactory epithelium.</a> J Comp Neurol. 483 (2), 154-163 (2005).</li><li>Cascella, N. G., Takaki, M., Lin, S., Sawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodevelopmental+involvement+in+schizophrenia:+the+olfactory+epithelium+as+an+alternative+model+for+research.">Neurodevelopmental involvement in schizophrenia: the olfactory epithelium as an alternative model for research.</a> J Neurochem. 102 (3), 587-594 (2007).</li><li>Sawa, A., Cascella, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+olfactory+system+for+clinical+and+basic+psychiatry:+a+promising+entry+point+to+the+mystery+of+brain+mechanism+and+biomarker+identification+in+schizophrenia.">Peripheral olfactory system for clinical and basic psychiatry: a promising entry point to the mystery of brain mechanism and biomarker identification in schizophrenia.</a> Am J Psychiatry. 166 (2), 137-139 (2009).</li><li>Mor, E., 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=MicroRNA-382+expression+is+elevated+in+the+olfactory+neuroepithelium+of+schizophrenia+patients.">MicroRNA-382 expression is elevated in the olfactory neuroepithelium of schizophrenia patients.</a> Neurobiol Dis. 55, 1-10 (2013).</li><li>Kano, 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=Genome-wide+profiling+of+multiple+histone+methylations+in+olfactory+cells:+further+implications+for+cellular+susceptibility+to+oxidative+stress+in+schizophrenia.">Genome-wide profiling of multiple histone methylations in olfactory cells: further implications for cellular susceptibility to oxidative stress in schizophrenia.</a> Mol Psychiatry. 18 (7), 740-742 (2013).</li><li>Toritsuka, 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=Deficits+in+microRNA-mediated+Cxcr4/Cxcl12+signaling+in+neurodevelopmental+deficits+in+a+22q11+deletion+syndrome+mouse+model.">Deficits in microRNA-mediated Cxcr4/Cxcl12 signaling in neurodevelopmental deficits in a 22q11 deletion syndrome mouse model.</a> Proc Natl Acad Sci U S A. 110 (43), 17552-17557 (2013).</li><li>Evgrafov, O. V., 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=Olfactory+neuroepithelium-derived+neural+progenitor+cells+as+a+model+system+for+investigating+the+molecular+mechanisms+of+neuropsychiatric+disorders.">Olfactory neuroepithelium-derived neural progenitor cells as a model system for investigating the molecular mechanisms of neuropsychiatric disorders.</a> Psychiatr Genet. 21 (5), 217-228 (2011).</li><li>Fan, 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=Focal+adhesion+dynamics+are+altered+in+schizophrenia.">Focal adhesion dynamics are altered in schizophrenia.</a> Biol Psychiatry. 74 (6), 418-426 (2013).</li><li>Ishizuka, 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=Negative+symptoms+of+schizophrenia+correlate+with+impairment+on+the+University+of+Pennsylvania+smell+identification+test.">Negative symptoms of schizophrenia correlate with impairment on the University of Pennsylvania smell identification test.</a> Neurosci Res. 66, 106-110 (2010).</li><li>Sattler, R., 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=Human+nasal+olfactory+epithelium+as+a+dynamic+marker+for+CNS+therapy+development.">Human nasal olfactory epithelium as a dynamic marker for CNS therapy development.</a> Exp Neurol. 232 (2), 203-211 (2011).</li><li>Rimbault, M., Robin, S., Vaysse, A., Galibert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+profiles+of+rat+olfactory+epithelia:+individual+and+age+related+variations.">RNA profiles of rat olfactory epithelia: individual and age related variations.</a> BMC Genomics. 10, 572 (2009).</li><li>Tajinda, 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+biomarkers+from+patients+with+mental+illnesses:+a+novel+method+through+nasal+biopsy+combined+with+laser-captured+microdissection.">Neuronal biomarkers from patients with mental illnesses: a novel method through nasal biopsy combined with laser-captured microdissection.</a> Mol Psychiatry. 15 (3), 231-232 (2010).</li><li>Bonner, R. 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278 (5342), 1481-1483 (1997).</li><li>Fink, 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=Real-time+quantitative+RT-PCR+after+laser-assisted+cell+picking.">Real-time quantitative RT-PCR after laser-assisted cell picking.</a> Nat Med. 4 (11), 1329-1333 (1998).</li><li>Emmert-Buck, M. R., 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=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274 (5289), 998-1001 (1996).</li><li>Nurnberger, J. 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=Diagnostic+interview+for+genetic+studies.+Rationale,+unique+features,+and+training.">Diagnostic interview for genetic studies. Rationale, unique features, and training.</a> NIMH Genetics Initiative. Arch Gen Psychiatry. 51 (11), 849-859 (1994).</li><li>Montgomery, S. A., Asberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+depression+scale+designed+to+be+sensitive+to+change.">A new depression scale designed to be sensitive to change.</a> Br J Psychiatry. 134, 382-389 (1979).</li><li>Young, R. C., Biggs, J. T., Ziegler, V. E., Meyer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rating+scale+for+mania:+reliability,+validity+and+sensitivity.">A rating scale for mania: reliability, validity and sensitivity.</a> Br J Psychiatry. 133, 429-435 (1978).</li></ol>

저자는 상업적 이익과 재정적 관계를보고하지 않습니다.

코 조직 검사 및 LCM을 결합하여 풍부한 후각 신경 층을 얻기위한 새로운 플랫폼을 제시하고 본 연구에서 검증되었습니다. 이 기술은 광범위하게 영향을 미칠 수 있습니다. 이 분야에서 폭 넓은 영향을, 치료 반응을위한 것들을 포함 바이오 마커 연구, 및 다른 조건에 대한 신경 정신병 약물 발견 노력으로 적용될 수있다. 다운 스트림 응용 프로그램에 대한 의무 고품질의 신?...

양극성 장애 (BD)이 기분 병리학 적 변화를 특징으로하는 심각한 신경 장애이며, 드라이브 및인지 1. BD의 치료에 사용되는 리튬은 동물 연구에서 두 유전자의 다수의 정상 상태의 mRNA 수준을 변경하기 위해 도시되었지만, 이들 분자 중 2 인간 임상 반응과 연관된 경우에는 알려지지 않았다. 리튬의 반응 메카니즘을 이해하는 것은 신경 조직에서의 리튬 - 유발 분자 변화를 조사 요구한다. 불행히도, BD 환자의 뇌 생검 전후 리튬 요법 리튬 반응 분자 서명을 식별하기 위해 수득 실용적이지 않다. 사후 뇌 조직은 BD 바이오 마커의 연구를 위해 사용 된, 그러나, 감정,인지와 이용시의 동적 변화와 관련 분자 마커를 평가하기 위해 사용될 수 없다; 소급 확인 리튬 치료 반응의 유효성이 문제가 될 수있다 (4). 림프구 및 기타 혈액 세포가 유용 할 수 있지만, 혈액 세포의 분자 변화는 신경 세포의 변화 3-5 반영되지 않을 수 있습니다. 뇌척수액은 질환 관련 약물 가역 본질적인 변화를 반영 할 수있다 세포 내 분자에 대한 정보를 얻을 불충분 할 수있다. 후각 상피 (OE)가 중추 신경계 (CNS)의 고유 부분이며, 변연 구조 (6)에 발생 학적으로 관련; 그리고 코 조직 검사를 통해 쉽게 액세스 할 수 있습니다. 그것은 이루어져 신경교 형상 개발 7-9의 다른 단계에서 (즉, sustentacular) 세포, 기저 세포 증식, 후각 수용체 뉴런지지. 따라서, OE는 접근 가능 신경 정신 질환 (7) 환자의 CNS의 역동적 인 변화를 연구하는 독특한 기회를 제공합니다. 연구는 그 OCC을 반영 질병 관련 사건을 조사하기위한 대리 조직으로 OE의 유용성을 입증하고 있습니다뇌 신경 세포 8,9에 urring. 예를 들어, 연구는 정신 상태 10-14과 관련된 분자 프로파일을 조사하기 위해 OE를 활용했다. 후각 시스템은 또한 정신 분열증 (15)의 음성 증상과 관련된 냄새 적자 임상 endophenoytpes을 식별하는 데 도움이된다. 또한, 신경 발달 과정은 정신 조건 8,9의 기본 병태 생리를 모델링 할 수있는 유용한 수단을 제공하고, 전 생애에 걸쳐 OE에서 계속합니다. 그러나,이 조직의 사용의 단점은 비 - 신경 세포와 함께 16 후각 생검의 실질적인 오염이다. 예를 들어, 이전의 연구에서 OE 유전자 발현 연구에 사용 된 총 RNA 비 신경 세포 (17)로부터 전체 RNA를 포함 비강 생검 조직에서 추출한 RNA를 함유 하였다. 따라서, 이전의 접근법은 세포의 품질에 의해 제한되어왔다. 이 문제를 극복하기 위해 새로운 접근 방식을 구하는<html> 레이저 캡처 미세 절제 (LCM)와 비강 조직 검사를 결합하여 풍부한 신경 세포 집단은 18 개발되었습니다. LCM은 적외선 레이저와 함께 19-21 UV 레이저 절삭을 사용하여 세포의 선택적 분리를 허용하는 기술이다. OE 방식과 결합 LCM함으로써 신경 세포 (18)의 농축을 강화, 비 신경 세포에 의해 OE 상당한 오염을 최소화 할 수 있습니다. 또한, 신경 층함으로써 염색에 대한 필요성을 제거 현미경 점막하 층으로부터 구별 될 수있다. 신경 세포 유형은 또한 관심 7 세포 유형에 의해 발현되는 일차 항체를 사용하여 다른 세포 집단 구별 될 수있다. 따라서,이 절차는 유전자 발현 연구, 면역 및 기타 형태 학적 연구에 사용할 수있는 거의 순수한 신경 세포 집단의 농축을위한 쉬운 방법을 확립한다. ve_content는 &quot;&gt;이 연구는 질병 상태와 치료 반응과 관련된 후각 신경 세포의 분자 변화를 조사하기 위해 실험 플랫폼을 구축하는 것을 목표로하고있다.이 문제를 해결하기 위해, BD의 DSM-IV 진단 기준을 충족 금연 환자의 작은 세트에 기반 유전 연구에 대한 진단 인터뷰 (DIG) (22)는 두 개의 코 조직 검사를 받아야하는 채용되었다 : 리튬과 두 번째 생검 한 조직 검사 전처리, 경구 리튬 치료 6 주 후에 또한, 자격 BD 환자 수 있어야합니다. 우울증 증상 몽고메리 - ASBERG 평가 척도 (MADRS) (23) 투여 임상의 60에서 ≥10 득점에 따라 (YMRS) 경조증 또는 조증 증상, 젊은 매니아 평가 척도 투여 임상에 56 점 만점 점수 ≥10에 따라 . 24;. MADRS 모두 저울에 대한 임상의 간의 합의에의 YMRS있는 Rater-간 레이터 계수 모두 또는 ≥10는 조직 검사 후 신경 enri 0.96했다&gt;는LCM에 의해 OE에서 CHED. 신경 조직 및 농축로부터 고품질의 RNA 추출을 위해, 추가의 품질 관리 방법에 따라 실시간 RT PCR은 관심 유전자의 사전 및 사후 처리의 발현 수준을 조사하기 위해 실시되었다. 다음의 섹션은이 방법의 유효성 검사에 대한 설명, 프로토콜의 최적화를 강조 표시하고 문제 촬영 프로토콜을 적용 된 전략이 포함되어 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Reagent</td>
			<td style="width:143px;">Manufacturuer&#160;</td>
			<td style="width:175px;">Manufacturer Catalog #</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek Cryomold Molds</td>
			<td>Sakura Finetek</td>
			<td>4557</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Tek O.C.T Compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryosectioning</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Membrane Slide 1.0 PEN (D)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>415190-9041-000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rnase Zap</td>
			<td>Ambion</td>
			<td>AM9780</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEPC Treated Water</td>
			<td>Quality Biological</td>
			<td>351-068-131</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microdissection</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope: PALM Series MicroLaser System</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Model: Axiovert 200M</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software: Robo v3.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">No.5 Dumont Microdissction Forceps</td>
			<td>Roboz</td>
			<td>RS-49085</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous Micro Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cDNA Synthesis</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III First Strand Synthesis Kit</td>
			<td>Invitrogen&#160;</td>
			<td>18080-051</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OMP qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR GreenER qPCR SuperMix</td>
			<td>Invitrogen&#160;</td>
			<td>11760-500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman qPCR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Expression Assay Probes</td>
			<td>Applied Biosystems</td>
			<td>Various</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
		</tr>
	</tbody>
</table>

olfactory neurons obtained through nasal biopsy combined with laser-capture microdissection: a potential approach to study treatment response in mental disorders

양극성 장애 (BD)이 제대로 이해 병태 생리학과 심한 신경 정신 질환과 전형적 기분 안정제, 탄산 리튬으로 처리 하였다. 동물 연구뿐만 아니라 인간의 유전 연구는 리튬이 신경 세포의 성장, 생존과 성숙, 그리고 Wnt 신호에 관여 특히 분자에 관여하는 분자 표적에 영향을 미친다는 사실을 보여. 중추 신경계 (CNS)에서 리튬 응답과 연관된 동적 분자량 변화를 조사하는 뇌 생검 구하기 윤리적 도전 감안할 번이 goal.The 후각 상피 후각 수용체 뉴런을 포함 달성 후각 조직으로부터 수득 된 뉴런의 사용을 고려할 수있다 개발 및 신경교 유사 세포를지지하는 다른 단계에서. 이 안전하게 코 조직 검사에서 얻은 후각 조직을 사용, 신경 정신 질환을 가진 환자의 CNS의 역동적 인 변화를 연구하는 독특한 기회를 제공합니다. SUBST로 인한 단점을 극복하기 위해비 신경 세포와 후각 생검 조직의 antial 오염, 풍부한 신경 세포 인구를 얻을 수있는 새로운 접근 방식은 레이저 캡처 미세 절제와 코 생검을 결합하여 개발되었다. 이 연구에서, 신경 조직에 치료 - 관련 분자의 동적 변화를 조사하는 시스템은 리튬 치료 반응의 분자 메커니즘의 연구를 위해 모집 BD 환자의 작은 파일럿 샘플을 사용하여, 개발 및 검증 하였다.

분자 서명의 연구에서 프로토콜 및 문제 해결 전략이 성공적으로 최적화되었다. RNA 샘플을위한 품질 및 신경 농축 기준 하류 리얼 타임 PCR 분석에 사용되는 표준화 하였다. RIN 및 RNA의 농도는 검출 된 발현 수준을 교란 요인으로 조사 하였다. 1에서부터 RINs 여러 샘플의 분석을 바탕으로 - (10)는, 상술의 ~ 3.0의 최소 RIN은 본 연구에서 분석 하류에 충분하다고 판단되었다. 결과적으로, cDNA의 합성을?...

Watch this Scientific Journal Video about Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disord

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

본 연구에서는 새로운 플랫폼을 개발하고 검증 된 양극성 장애 (BD)의 치료 반응의 intraneuronal 분자 서명을 조사합니다. BD 환자의 후각 상피 비강 생검을 통해 얻었다. 이어서 레이저 캡처 미세 절제가 BD 리튬 응답의 분자 특성을 조사하기 위해 실시간 RT PCR과 결합시켰다.

레이저 캡처 이외에 Microdissection과 코 생검 결합을 통해 획득 후각 뉴런 : 정신 장애의 치료 반응을 연구하기 위해 잠재적 인 접근

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

olfactory-neurons-obtained-through-nasal-biopsy-combined-with-laser

Research

JoVE Journal

Neuroscience

9.7K 조회수.  Johns Hopkins University.  본 연구에서는 새로운 플랫폼을 개발하고 검증 된 양극성 장애 (BD)의 치료 반응의 intraneuronal 분자 서명을 조사합니다. BD 환자의 후각 상피 비강 생검을 통해 얻었다. 이어서 레이저 캡처 미세 절제가 BD 리튬 응답의 분자 특성을 조사하기 위해 실시간 RT PCR과 결합시켰다. 

비디오: Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

의 레이저 캡처 Microdissection Drosophila 주변 뉴런

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

연구

신경과학

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

설치류 또는 인간의 네이절 강한 줄기 세포를 분리

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

결합을 사용하여 타겟팅 후각 망울의 뉴런 VIVO에서 Electroporation 및 Gal4 기반 향상제 트랩 Zebrafish 라인

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

총 RNA의 분리 및 다운 스트림 차세대 시퀀싱 및 유전자 발현 프로 파일링에 대한 개별 마우스 뇌 영역의 Microdissection 아닌 레이저 캡처 현미경 접근법

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

외상성 뇌 손상 후 뉴런 또는 유전자 발현 분석을위한 단일 뉴런의 강화 인구의 레이저 캡처 Microdissection

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

문화의 차별화 된 인간 Neuroprogenitor 세포에서 신경 세포의 레이저 캡처 미세 절제

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

레이저 포획 미세 절에 의해 마우스 척수 모터 신경 세포 체에서 전사 분석을위한 RNA의 생산

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

레이저 캡처 이외에 Microdissection - 개별 도파민 신경 세포의 분리 및 전체 복부 피개 영역의 데모

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

외상 성 부상에 의해 영향을 하는 뇌 영역의 정위 적 아틀라스 유도 레이저 캡처 기정

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.