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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1, Culture Media (50 ml)</td>
		</tr>
		<tr>
			<td>Neurobasal&#160;</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></td>
			<td></td>
			<td></td>
			<td>2, washing media (HBSS, 100 ml)</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red&#160;</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></td>
			<td></td>
			<td></td>
			<td>3, HEPES buffered saline&#160; (HBS)&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4, HBS Cocktail for live imaging pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>tetrodotoxin&#160;</td>
			<td>Tocris</td>
			<td>1078</td>
			<td>2 &#181;M</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Tocris</td>
			<td>131</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td>D-AP-5</td>
			<td>Tocris</td>
			<td>105</td>
			<td>50 &#181;M</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>1045</td>
			<td>20 &#181;M</td>
		</tr>
		<tr>
			<td>LY341495</td>
			<td>Tocris</td>
			<td>1209</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>5, Calcium-free&#160; HBS&#160;&#160; pH=7.3</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888&#160;</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>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&#160;</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 ...

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Unit 1

Drugs Acting on Autonomic Nervous System: Adrenergic Agonists and Antagonists Agents

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8.9K 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

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation 및 Mitochondrial 교통 : 긴 기간이 이상 Hippocampal 뉴런의 라이브 영상

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

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신경과학

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Rhizotomy 후 도설 루트 Axons의 라이브 영상

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

칼슘 이미징에 대한 급성 Subventricular 지대 슬라이스의 작성

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

성장 Axons 함께 손쥐 Schwann 세포 개발 분석

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

차 해마 신경 세포의 칼슘 인산염 형질

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

의 라이브 영상<em&gt; 초파리</em&gt; 애벌레 Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

신경 전달 물질 방출 공부 하 느 루트 중추 절연 및 주 문화

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

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 ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.