We thank Yehui Qin and Mallory Myers for technical support. We also thank Dr. Sigismund Huck for providing us the anti-&#945;4-ECD antibody. This work is supported by National Institutes of Health grant NS22061 to L. W. R.

모든 동물 실험 (2012 개정, NIH 간행물 번호 80-23) 실험 동물의 관리 및 사용을위한 건강 가이드의 국립 연구소에 따라 수행되었고, 연구 기관 동물 관리의 승인을 스토니에 연구위원회에 사용되었다 브룩 대학 (# 1618과 # 1792). 1. vHipp-NACC 시냅틱 공동 문화 <ol><li> 희생 마우스 (출생 후의 일 0 - 야생형 (WT) 또는 α7 nAChRs 유전자 변형 마우스 라인 3) CO 2. 각각의 강아지 목을 벨 및 사후 사실상의 유전자형에 대한 1.5 ml의 원심 분리기 튜브에 꼬리를 저장합니다. 멸균 후드에서 다음 단계를 수행합니다. </li><li> 전체 뇌를 노출 가위와 집게로 두개골의 상단을 제거합니다. 서로 꼬리 쪽의 대뇌 피질 부분은, 해부 현미경 vHipp 5,21 등 후방 피질을 포함하는 코로나 섹션을 얻을 시점에서 시작. </li><li> V를 해부하다개발 마우스 뇌 아틀라스 (22) (그림 1A)에 따라 CA1-subiculum 지역을 포함 HIPP 조직을 제거합니다. 추운 문화 미디어 (4 ° C의)를 포함하는 35mm 배양 접시로 전송, 작은 얇은 조각 (μm의 약 100 × 100 μm의 microslices)으로 잘라. </li><li> 37 ° C의 배양기에서 적어도 30 분 동안 배지 (~ 500 μL)를 24 웰 조직 배양 플레이트 내부 12mm 폴리 D 라이신 / 라미닌 - 코팅 유리 커버 슬립을 사전 부화. microslices 도금하기 전에 용지를 제거합니다. </li><li> (microslices의 ~ 20 개 함유 ~ 50 μL) 미디어의 최소량과 커버 슬립의 중심에서 화재 연마 파스퇴르 피펫 접시는 이식편의 부착을 용이하게한다. 하나의 동물에서 발생하는 플레이트 vHipp의 microslices (중 + / + 또는 - / - 유전자 변형 α7 라인의 유전자형) 각 coverslip에에. </li><li> 외식은 커버 슬립에 정착 한 후, 부드럽게 일까지 추가 미디어 (~ 100 μL)를 추가E 벽되도록 매체의 레벨은 아니지만 그 커버 슬립의 중앙에서 완전히 둘레에 이식편을 커버하기에 충분히 높다. </li><li> 37 ° C에서 O / N 배양 한 후, 5 % CO 2 배양기, 출생 후 1 일 (WT) 쥐에 배아 일 18 NACC 신경을 분산하고 외식에 추가 할 수 있습니다. <ol><li> 개발 마우스 뇌 아틀라스 (20) (그림 1B)에 따라 NACC 조직을 해부하다. </li><li> 15 ML 튜브 및 전송 (500 500 μm의 microslices × μm의 주위에) 작은 조각으로 잘라. </li><li> 37 ° C에서 15 분 동안 0.25 % 트립신 (2 ㎖)로 처리. 워시 조직 덩어리를 차가운 세척 미디어 3 회 (5 ㎖, 4 ° C의) 차가운 문화 매체에서 한 세척 한 다음 (5 ㎖, 4 ℃). 서스펜션은 각 세척 단계에서 5 분간 휴식 한 다음 조심스럽게 상층 액을 부어하도록 허용합니다. </li><li> 가볍게 화재 광택 파스퇴르 피펫으로 배양 배지 2 ㎖ 부드러운 분쇄하여 세포를 떼어 놓다. </li><li> 트랜스5 분 동안 2,000 X rpm에서 또 다른 15 ML 튜브와 원심 분리기에 FER 세포 현탁액. 상층 액을 제거하고 6 새끼 당 1 ml의에서 배양 배지에서 세포를 재현 탁. 부드럽게 화재 연마 파스퇴르 피펫으로 언론에 여러 번 피펫 팅하여 세포를 분산. </li><li> vHipp 외식 각 coverslip에에 분산 NACC 세포의 0.25 ML을 추가합니다. </li><li> 가습 37 ° C의에서 문화를 유지, 5 % CO 2 배양기. 기계적으로 시냅스 형성을 촉진, 안정 및 습도를 유지하기 위해 적신 멸균 거즈 패드에 문화 판을 놓습니다. </li></ol></li></ol> 2. 면역 세포 화학 <ol><li> 문화 수정 - 4 % 파라 포름 알데히드 / 4 %의 자당 (5 체외에서 7 일) / PBS (~ coverslip에 500 μL, 20 분, RT). </li><li> 0.25 % 트리톤 X-100 / PBS와 Permeabilize 하시려면 배양 (~ 커버 슬립 당 ​​500 μL, 5 분, RT). </li><li> PBS 10 % 정상 당나귀 혈청 블록 문화 (~ coverslip에 500 μL, 30 분, RT) antibod 이전Y 염색. 4 ° C에서 O / N 일차 항체에 품어. 안티 포성 글루타메이트 트랜스 포터 1 (1 : 250), 안티 GAD65 (1 : 100); 안티 nAChRs (항 α 5 소단위, 1 500 500 α 4 -ECD 1) 다음과 같은 일차 항체를 사용합니다. </li><li> (coverslip에 3 회 / ~ 500 μL, 5 분) PBS로 일차 항체를 세척 한 후 (~ coverslip에 500 μL, 1 : 500) 알렉사 488에 복합 이차 항체에 문화를 품어 또는 커버 슬립 당 ​​알렉사 594 (~ 500 μl를 1 : 37 ℃에서 1 시간 동안 500). </li><li> 이전 라벨 표면 α7 * nAChRs에 대한 고정 37 ° C에서 45 분 (문화 미디어에서 1,000 ~ coverslip에 500 μL, 1) 알렉사 594에 결합 αBgTx의 문화를 품어. </li><li> 마운트 및 밀봉 된 커버. </li><li> 플랜-고차 색지움 목표 (0.8 NA 1.4 또는 NA와 오일 63X 20X)을 구비 한 형광 현미경과 CCD 카메라로 이미지를 캡처. </li></ol> 3. FM1 -​​ 43 기반 소낭 퓨전 <ol><li> , 영상 실 - (체외에서 7 일 5) 디스크 공 초점 현미경을 회전에 챔버를 탑재 문화를 유지한다. 지속적으로 실온에서 HBS 칵테일 (1 ml / 분)를 관류. </li><li> 90 초 동안 56 mM의 K + ACSF (~ 500 μL) (염색)에 10 μm의 FM1 - 43와 관류 및로드 문화를 중지, 칼슘 ADVASEP-7 (0.1 mm)를 포함하는 무료 HBS로 15 분 거리에 외부 염료를 씻어 막 결합 FM1 -​​ 43을 청소할 수 있습니다. </li><li> 120 초 동안 FM1 - 43없이 56 mM의 K + ACSF (~ 500 μL) (탈색)과 문화를 자극 같은 조건을 사용하여 FM1 - 43와 문화를 다시로드, 및 CA 다시 포함 2+ 무료 HBS 씻어 ADVASEP-7 15 분. </li><li> CCD 카메라와 계획 - 고차 색지움 목표 (1.4 NA와 60X 물, 여기 488 nm의, 방출 53​​0 ㎚)과 캡처 이미지를 갖춘 회전 디스크 공 초점 현미경으로 FM1 -​​ 43 형광 이미지를 획득. </li><li> 1 분 기준 FM1 -​​ 43 형광 이미지 (매 2의 수집) 사전 니코틴 제어와 같은; 1 분 동안 빠른 관류 (2 ml / 분)에 의해 니코틴 (1 μM)를 적용하고 밖으로 HBS 칵테일 니코틴을 씻어. 5 분 동안 매 2 초 동안, 니코틴 신청 후, 전에 캡처 시간 경과 이미지를 유지합니다. </li><li> 전에 FM1 - 43 형광 강도 (F 염색)과 후 (F의 탈색) 각 시냅스의 부통에서 니코틴 응용 프로그램을 정량화. </li><li> 이전과 니코틴 응용 프로그램 (ΔF = F 염색 -F의 탈색) 후 FM1 - 43 형광 강도의 차이를 정량화하여 vHipp 축삭을 따라 해제 FM1 - 43 형광의 총량을 계산합니다. 다음 식에 니코틴 후 형광 강도 저하의 비율을 분석 : F 감소 % = ΔF / F 염색. </li></ol> 4. 칼슘 이미징 <ol><li> 다음 (5 μm의 FLUO-4 칼슘 2 + 지표와 문화를로드, 정상 HBS 빠르게 - (체외에서 7 일 5) 문화를 씻어AM 에스테르) 및 37 ° C에서 30 분, 5 % CO 2의 HBS 0.02 % 플루로 닉 F-127 (2 mL)을 첨가 하였다. </li><li> (3 회 / 5 분) HBS와 FLUO-4 솔루션을 세척 할 것. (37 ° C / 5 % CO 2) 적어도 30 분 동안 인큐베이터 문화를 돌려줍니다. </li><li> 동일한 촬상 실과 FM1 -​​ 43 기반 소포 융합 절에서 설명한 바와 같이 동일한 조건으로 배양 물을 유지한다 (단계 3.1 참조). </li><li> 계획 - 고차 색지움 목표 (1.4 NA와 60X 물, 여기 488 nm의, 방출 53​​0 ㎚)와 CCD 카메라와 vHipp 마이크로 조각에서 축삭 돌기의 FLUO-4 형광 이미지를 수집합니다. </li><li> 1 분 동안 빠른 관류 (2 ml / 분)에 의해 니코틴 (1 μM)를 적용, 사전 니코틴 컨트롤로 기준 FLUO-4 형광 이미지 (2 분마다 10 초)을 수집 한 다음 밖으로 HBS 칵테일 니코틴을 씻어. 30 분마다 10 초 동안, 니코틴 신청 후, 전에 캡처 시간 경과 이미지를 유지합니다. </li><li> 일련의 원시 FLUO-4 형광 이미지, 수출의 모든 프레임을 저장추가 분석을 위해 TIFF 포맷 이미지. </li><li> 전에 vHipp 축삭을 따라 니코틴 신청 후 FLUO-4 형광의 통합 강도를 수집합니다. </li><li> 하기 식으로 통합 된 형광 강도를 정규화한다 ΔF / F = 0 (FF 0) / F 0 배경 보정 전 니코틴 집적 형광 강도이며, F는 각각의 시점에서 vHipp 축삭을 따라 집적 형광 강도 F 0, 니코틴 신청 후. 분석하고 정규화 집적 형광 강도를 플롯. </li></ol>

<ol>
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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=Trafficking+of+alpha4*+nicotinic+receptors+revealed+by+superecliptic+phluorin:+effects+of+a+beta4+amyotrophic+lateral+sclerosis-associated+mutation+and+chronic+exposure+to+nicotine.">Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.</a> J Biol Chem. 286 (36), 31241-31249 (2011).</li><li>Colombo, S. F., Mazzo, F., Pistillo, F., Gotti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+trafficking+and+up-regulation+of+nicotinic+ACh+receptors.">Biogenesis, trafficking and up-regulation of nicotinic ACh receptors.</a> Biochem Pharmacol. 86 (8), 1063-1073 (2013).</li><li>St John, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+trafficking+of+nicotinic+acetylcholine+receptors.">Cellular trafficking of nicotinic acetylcholine receptors.</a> Acta Pharmacol Sin. 30 (6), 656-662 (2009).</li><li>Zhong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+type+III+neuregulin+1+is+required+for+sustained+enhancement+of+hippocampal+transmission+by+nicotine+and+for+axonal+targeting+of+alpha7+nicotinic+acetylcholine+receptors.">Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors.</a> J Neurosci. 28 (37), 9111-9116 (2008).</li><li>Jacobowitz, D. M., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemoarchitectonic Atlas of the Developing Mouse Brain. , (1998).</li><li>Betz, W. J., Mao, F., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+fluorescent+staining+and+destaining+of+living+vertebrate+motor+nerve+terminals.">Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.</a> J Neurosci. 12 (2), 363-375 (1992).</li><li>Amaral, E., Guatimosim, S., Guatimosim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+fluorescent+styryl+dye+FM1-43+to+visualize+synaptic+vesicles+exocytosis+and+endocytosis+in+motor+nerve+terminals.">Using the fluorescent styryl dye FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor nerve terminals.</a> Methods Mol Biol. 689, 137-148 (2011).</li><li>Garduno, 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=Presynaptic+alpha4beta2+nicotinic+acetylcholine+receptors+increase+glutamate+release+and+serotonin+neuron+excitability+in+the+dorsal+raphe+nucleus.">Presynaptic alpha4beta2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.</a> J Neurosci. 32 (43), 15148-15157 (2012).</li><li>Guo, J. Z., Liu, Y., Sorenson, E. M., Chiappinelli, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+and+exogenous+ACh+activates+different+nicotinic+receptors+to+enhance+evoked+glutamatergic+transmission+in+the+lateral+geniculate+nucleus.">Synaptically released and exogenous ACh activates different nicotinic receptors to enhance evoked glutamatergic transmission in the lateral geniculate nucleus.</a> J Neurophysiol. 94 (4), 2549-2560 (2005).</li><li>Szabo, S. I., Zelles, T., Vizi, E. S., Lendvai, 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+effect+of+nicotine+on+spiking+activity+and+Ca2++dynamics+of+dendritic+spines+in+rat+CA1+pyramidal+neurons.">The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons.</a> Hippocampus. 18 (4), 376-385 (2008).</li></ol>

저자가 공개하는 게 없다.

공동 배양 제제는 기재된 재 저항하는 것을 시험 관내 복부 해마-중격 의지 회로. 프리 시냅스 nAChRs의 활성화는 글루타메이트 변속기 (5), (21)를 강화 유도하는 공간 및 시간 프로파일이 제조 허가 비교적 간단하고 신뢰성 시험. 공동 배양 생체 -like 함수를 보여주는 시험 관내에서 생리적 조건을 제공 할 수있는 하나의 접시에 다른 특정 세?...

회로 흥분성의 콜린성 변조인지의 기본적인 양태에 기여하고, 변경된 콜린성 변조는 알츠하이머 병, 파킨슨 병, 정신 분열증 및 중독 1-4 포함한 신경 퇴행성 질환 및 신경 기능이다. 중추 신경계의 시냅스 전달의 콜린성 촉진의 설립 메커니즘은 사전 시냅스 사이트에서 번역 된 nAChRs 직접 활성화를 통해입니다. 간접적으로 특정 nAChR을 서브 타입, 그리고 상대적으로 높은 칼슘 전도에 직접적으로 인해를 통해 세포 내 신호 폭포 -이 사전 시냅스 수용체의 활성화는 세포 내 칼슘 ([칼슘 2 +] I) 전 시냅스 터미널의 증가에 이르게 5함으로써 신경 전달 물질 방출을 향상. 실제로, 프리 시냅스 nAChRs의 활성화는 글루타메이트 신경 전달 물질을 포함한 다양한, GABA, 아세틸 콜린의 방출의 변화와 연관되어있다차 도파민 6-10. 이 프로세스는 다양한 간접적 시냅스 전기 생리 학적 방법을 이용하여 검토했지만, 광학 기자 [CA는 2+] i 및 시냅스 소포 재활용 프리 시냅스 현상의 직접적인 시간적으로 정확한 측정을 허용한다. nAChRs의 사전 시냅스 현지화는 전자 현미경 (EM) 레벨 11, 12에 nAChRs 직접 면역 금 표지와 함께 설득력있게 입증되었다. 다른 여러 기술들은 배양 된 신경 세포 (13, 14)에 nAChRs subunit- 형광 단백질 키메라의 위치를 검출 포함한 간접적 nAChR을 현지화를 해결하기 위해 사용되고, 시냅스 말단 (15, 16)에 nAChR을 전류의 전기 생리 학적 기록 [캘리포니아 니코틴 유도 변화를 모니터링 에 의해 시냅스 터미널에서 라이브 세포 이미징 (17), 및 신경 전달 물질 방출의 간접적 인 모니터링에 의한 시냅스 신경 터미널에서 2 +] I스티 릴 양친 매성 FM 염료 (FM1 -​​ 43와 FM4-64) 및 / 또는 synapto-pHluorin에 의해 및 글루타메이트에 대한 아세틸 콜린과 iGluSnFr에 대한 CNiFERs 같은 특정 형광 신경 전달 물질 기자, 볼 시냅스 소포의 세포 외 유출을 포함 형광 지표와 라​​이브 세포 이미징 기법, 18 ~ 20. 전반적으로, nAChRs의 사전 시냅스 현지화를 식별이 현재의 접근 방법이 복잡하고, 신뢰할 수있는 식별 및 사전 시냅스 활동의 생리 학적 모니터링을 할 수 있도록 특별한 시스템과 기술을 필요로한다. 확인하고 시냅스 전달의 사전 및 사후 시냅스 구성 요소를 모두 분석에 직접 액세스를 제공합니다 핵 중격 의지 (NACC) 회로 - 여기서 우리는 복부 해마 (vHipp)의 체외 공동 문화 시스템에 대한 프로토콜 및 장비에 대해 설명합니다. 우리는 전 시냅스 nAChRs의 현지화 및 칼슘 신호와 신경 전달 물질 방출 ALO 매개 nAChR을의 라이브 세포 이미징의 예를 보여NG vHipp 축삭. 여기에 제시된 프로토콜 천연 (및 간단한) 확장은 다른 유전자형으로부터 뉴런 구성된 전후 시냅스 접촉의 제조이다. 이러한 방식으로 변조 전 및 / 또는 후 - 시냅스 메커니즘 특정 유전자 산물의 기여는 직접 평가할 수있다.

<table><tbody><tr><td colspan="4">1. Culture Media (50 ml)</td></tr><tr><td>Neurobasal&amp;nbsp;</td><td>GIBCO</td><td>10888022</td><td>48 ml</td></tr><tr><td>B-27 Supplements</td><td>GIBCO</td><td>0080085-SA</td><td>1 ml</td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO</td><td>10908-010</td><td>0.5 ml</td></tr><tr><td>GlutaMAX Supplement</td><td>GIBCO</td><td>35050-061</td><td>0.5 ml</td></tr><tr><td>Brain-derived neurotrophic factor (BDNF)</td><td>GIBCO</td><td>15140-122</td><td>20 ng/ml</td></tr><tr><td colspan="4">2. washing media (HBSS, 100 ml)</td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red&amp;nbsp;</td><td>GIBCO</td><td>14175-095</td><td>99 ml</td></tr><tr><td>HEPES ( 1M)</td><td>GIBCO</td><td>15630-130</td><td>1 ml</td></tr><tr><td colspan="4">3. HEPES buffered saline&amp;nbsp; (HBS)&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">4. HBS Cocktail for live imaging pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>CaCl2,</td><td>Sigma</td><td>C1016&amp;nbsp;</td><td>2 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td>tetrodotoxin&amp;nbsp;</td><td>Tocris</td><td>1078</td><td>2 &amp;micro;M</td></tr><tr><td>bicuculline</td><td>Tocris</td><td>131</td><td>10 &amp;micro;M</td></tr><tr><td>D-AP-5</td><td>Tocris</td><td>105</td><td>50 &amp;micro;M</td></tr><tr><td>CNQX</td><td>Tocris</td><td>1045</td><td>20 &amp;micro;M</td></tr><tr><td>LY341495</td><td>Tocris</td><td>1209</td><td>10 &amp;micro;M</td></tr><tr><td colspan="4">5. Calcium-free&amp;nbsp; HBS&amp;nbsp;&amp;nbsp; pH=7.3</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888&amp;nbsp;</td><td>135 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333&amp;nbsp;</td><td>5 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M8266&amp;nbsp;</td><td>1 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375&amp;nbsp;</td><td>10 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr><tr><td colspan="4">6. 56 mM Potassium ACSF pH=7.4</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td>119 mM</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td>56 mM</td></tr><tr><td>MgSO4.7H</td><td>Sigma</td><td>M1880</td><td>1.3 mM</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C1016</td><td>2.5 mM</td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S8282</td><td>1 mM</td></tr><tr><td>NaHCO3</td><td>Sigma</td><td>S5761</td><td>26.2 mM</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G0350500&amp;nbsp;</td><td>10 mM</td></tr></tbody></table>

live imaging of nicotine induced calcium signaling and neurotransmitter release along ventral hippocampal axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

사용 준비는 체외에서 vHipp-NACC 회로의 유전자 키메라 공동 문화로 구성되어 있습니다. vHipp의 microslices에서 냄새가 나는데 계획은 사전 시냅스 축삭 입력으로, 후 시냅스 대상, NACC에서 분산 된 뉴런과 시냅스 연락처를 만들 수 있습니다. vHipp에 의해 신경 지배 NACC 신경 세포에서 글루타메이트 전송의 지속 (≥ 30 분) 촉진을 유도 니코틴은 사전 시냅스 α7 * nAChRs 통해 vHipp 축삭 (5)에 ?...

Watch this Scientific Journal Video about Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons at JoVE.com

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

니코틴 유도 칼슘 신호 전달 및 복부 해마 축삭을 따라 신경 전달 물질 방출의 라이브 영상

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

live-imaging-nicotine-induced-calcium-signaling-neurotransmitter

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9.0K 조회수.  Stony Brook University. We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

비디오: Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

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

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

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

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

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

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신경과학

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

만성 니코틴 관리와 마우스 노크 인의 Fluorescently 태그를 Nicotinic 수용체의 분광 공촛점 이미징

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

살아있는 마우스에 있는 등도 해마 CA1의 세로 2 광자 화상 진찰

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

행동분석학

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Photoactivatable 니코틴의 레이저 플래시 Photolysis를 통해 마우스 뇌 조각에서 Nicotinic 아 세 틸 콜린 수용 체 기능을 프로 빙

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

마우스를 면도하는 해마에서 신경 활동을 시각화하기위한 두개골 절제술 절차

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

생체 내 온전한 등근 신경절에 있는 일차 감각 뉴런 네트워크에서 뉴런 앙상블의 칼슘 이미징

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Co-culturing of Ventral Hippocampal and Nucleus Accumbens

Source: Zhong, C., et al. <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates the process of preparing ventral hippocampal and nucleus accumbens tissues from mouse brains for co-culture. Neurons extend projections to form synaptic connections between the ventral hippocampal and nucleus accumbens tissues. This method allows studying neuronal interactions between these two brain regions, offering insights into cognition, behavior, and neurological disorders.

Ventral Hippocampal 및 Nucleus Accumbens의 공동 배양

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Source: Zhong, C., et al., <a href="https://app.jove.com/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

시냅스 공동 배양에서 배쪽 해마 축삭을 따라 니코틴 유도 칼슘 신호 전달 이미징

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Calcium Imaging

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