지원은 의학의 피츠버그 대학의 학교에서 약리학 및 화학 생물학과에서 시작 - 기금에 의해 제공되었다. 니콜라스 그라프 : 비디오 제출에 기여 야곱 실험실 구성원의 확인.

아래에 설명 된 모든 프로토콜은 IACUC 의학의 피츠버그 대학의 대학의 IRB 제도 평가 보드에 따른다. 1. 조직 문화 후드에있는 해마 신경 세포 배양의 준비 주 : 1 프로토콜에 걸쳐 사용 멸균 기술과 시약. <ol><li> 신경 세포의 배양 기질로 폴리-D-라이신 (0.1 ㎎ / ㎖의 H 2 O) 코팅 된 유리 커버 슬립을 준비합니다. <ol><li> 각 3.5 cm 조직 배양 접시 안에 4-5 라운드 유리 커버 슬립을 놓습니다. </li><li> 각 12mm의 coverslip에 위에 자리 70 ㎕의 폴리-D-라이신. 참고 : 라이브 영상을 위해, 유리는 3.5 cm의 조직 배양 접시 (내장 14mm 유리 커버 슬립에 200 ㎕의 폴리-D-라이신 자리)를 사용할 수 있습니다 바닥. </li><li> 조직 문화 후드의 O / N.에 준비된 요리를 남겨 폴리-D-라이신 증발을 최소화하고 조직 문화 후드에서 멸균 표면을 유지, 창틀을 닫고 일을 해제하려면전자 송풍기 O / N. 참고 : UV 빛은 O / N 배양 중에 사용되지 않습니다. </li><li> 다음날 아침 씻어 요리 H 2 O 2 ㎖로 3 배 </li><li> 2 O 세척 마지막 H를 제거한 후, 각 3.5 cm 접시에 미디어 2 ㎖를 추가하고 단계 1.4에 신경을 도금 할 준비가 될 때까지 인큐베이터에서 접시를 둡니다. </li></ol></li><li> 해마 신경 세포는 배아 일 18-19 (Goslin 22에서 수정) (E18-19) 쥐에서 준비가되어 있습니다. </li><li> maxiprepped 구조의 DNA의 1-4 μg과 배양의 날에 갓 해리 신경 세포를 형질. 참고 : 일반적으로 DNA의 3 μg 50 %의 생존과 2 백만 신경 세포로 시작하는 60 %의 형질 전환 효율 (예 :와, 1-2000000 신경 세포로 형질 전환된다 : ((2 × 10 6 뉴런 × 0.5) 0.6)를 제공합니다 약 1 백만 신경;..의 잠재적 인 문제를 해결할 때 1을 형질 600,000있는 구조의 DNA의 다른 양의 형질 전환 될 수있다구조의 현지화 및 기능의 적절한 특성 다음에 표현. </li><li> 3.5 cm 접시 당 약 20 만 뉴런의 최종 밀도 폴리 - D - 라이신 코팅 커버 슬립에 형질 전환 된 뉴런을 접시. 참고 : 유리 접시를 바닥에, 14mm 유리 지역에 4 만 ~ 5 만 신경을 접시. </li><li> 미디어에게 연결을 문화의 준비 후 4-24 시간을 교체 한 후 뉴런은 체외 (DIV) 또는 원하는 단계에서 14~17일 때까지 인큐베이터에서 개발할 수 있습니다. </li></ol> 2. 엔도 시토 시스의 분석 주의 : α-bungarotoxin 및 tubocurarine 폐기물 및 취급시주의 사항 : <ol><li> 모든 펩타이드 신경 독소의 처리는 바이오 안전성 레벨 2 (BL-2) 연구 프로토콜의 가이드 라인에 좋습니다. 모든 적절한 개인 보호 장비를 착용해야한다. 동결 건조 독소 분말은 제조 업체의 지침에 따라 재구성해야한다. 독소 폐기물 수오LD는 통치 기관의 환경, 보건 및 안전 지침을 준수하여 폐기 될 수있다. </li><li> 저장 중에 형성 할 수있는 잠재적 인 펩티드 응집물을 제거하고, α-bungarotoxin 분취 량은 사용 전에 잠깐 원심 분리되어야하고, 상청액은 실험을 위해 사용되어야한다. </li><li> α-bungarotoxin (BGT) 주식은 -20 ℃에서 멸균 H 2 O 1 ㎎ / ㎖의 농도로 재현 탁하고 10 ㎕의 분취 량에 저장됩니다 형광 결합 BGT의 주식은 3 ㎍ / ml로 사용된다. Tubocurarine 150 μM의 최종 농도로 사용된다. </li></ol><ol><li> 위에 얇은 알루미늄이나 스테인리스 강판 차가운 방에서 16 ° C로 설정 벤치 탑 히터 블록. 가능한 경우 또는, 벤치 탑 냉각 / 가열 장치는 16 ° C를 필요로하는 각 단계에 사용할 수 있습니다 </li><li> 밀리미터에 포함 쿨 세포 헤 페스 완충 식염수 (HBS : 135의 NaCl, 4.7 KCl을, 10 HEPES, 11 포도당, 1.2 MgCl2를, 2.5염화칼슘, 물을 욕조에 16 ° C에 산도 7.4). </li><li> 16 ° C에서 알루미늄 판에 전송 신경 세포의 요리를 5 분 동안 차가운. </li><li> 용지를 제거하고 (단계 2.8, 엔도 시토 시스의 시간을 분석 포인트를 인큐베이터에서 에어컨 미디어 및 예비 유지) 2 분 동안 16 ° C HBS +의 tubocurarine (150 μM)의 1 ㎖로 대체합니다. </li><li> 라벨 표시 한 폐기 용기에 HBS + tubocurarine를 제거하고 알렉사 594 [3 ㎍ / ㎖의], 15 분 동안 16 ° C에서 배양 1 ㎖ 16 ° C HBS +의 tubocurarine (150 μM) + α-bungarotoxin로 교체. </li><li> 제거 HBS + tubocurarine + α-bungarotoxin 알렉사 594 (세척 50 % 표백제 50 ㎖를 포함하는 진공 플라스크에 흡입을 통해 수집 할 수 있습니다) 폐기물 컨테이너를 표시하고 요리를 16 ° C HBS의 2 ㎖로 3 배 세척합니다. </li><li> 유리를 사용하여 수용체 엔도 사이토 시스를 따르는 라이브 영상 시계열 실험은 3.5 cm 배양 접시 바닥의 경우, 단계 2.1-2.6를 수행 가열 단계로 요리를 전송(펠티어 소자) 현미경과 공 초점 또는 TIRF 현미경 설정으로 촬영을 시작한다. </li><li> 2.9 단계로 다른 커버 슬립을 계속, 20 분 동안 RT 4 % 파라 포름 알데히드 / 4 % 자당의 접시에 T = 0 시점의 coverslips을 전송합니다. </li><li> 다른 시간 지점의 경우와 HBS 교체 37 ° C 미디어 (단계 2.4에 예약) 및 37 ° C. 주에서 엔도 사이토 시스를위한 인큐베이터에 반환이 평형 에어컨 : 추가 시간 T = 15 점, 30, 60를 제안했다. </li><li> 시점에서 필요한, ​​인큐베이터에서 접시를 취할 RT DPBS 2 ㎖로 빠르게 두 번 세척 (더 칼슘, 마그네슘을 DPBS 없음) 한 후 20 분 동안 4 % 파라 포름 알데히드 / 4 %의 자당에 고정합니다. </li><li> 20 분 고정 후, 용기를 낭비하고 2 ㎖ RT DPBS로 두 번 설거지를 4 % 파라 포름 알데히드 / 4 %의 자당을 제거합니다. </li><li> 면역 형광 블록 용액에 10 분 동안 RT에서 커버 슬립을 배양한다 (블록 용액 = DPBS에 5 % 말 혈청, 0.5 % BSA) permeabil 위하여 0.2 % 트리톤 X-100을 함유신경이지는 세포 내 수용체 수영장 및 / 또는 관심의 다른 단백질의 라벨의 안티 GFP 면역 염색을 가능하게합니다. </li><li> 블록 + 0.2 % 트리톤 X-100을 제거하고 20 ~ 30 분 동안 트리톤 X-100하지 않고 면역 블록 솔루션의 coverslips을 품어. </li><li> 4 ℃에서 RT 또는 O / N에서 몇 시간 동안 블록에 커버 슬립의 기본 항체 배양을 수행 참고 : 4 ° CO / N에서 예비 선거와 커버 슬립의 배양이 일상적으로 향상된 면역 결과를 얻을 수 있습니다. </li><li> 워시 DPBS (각 세척 단계에 대한 5 분)와 함께 배를 커버 슬립. </li><li> 실온에서 1 시간 동안 차단 솔루션 차 항체와의 coverslips을 품어. </li><li> 워시 DPBS (각 세척 단계에 대한 5 분)와 함께 배를 커버 슬립. </li><li> 개별적으로 커버 슬립을 처리 마운트 커버 슬립이 : 실험실 조직과 커버 슬립의 뒷면에서 물기를 제거한 다음 유리 슬라이드에 장착 매체의 4 μL에 커버 슬립을 반전. </li><li> 실온에서 건조 슬라이드 생 후 30 분 동안 적용 허용4 ° C에서 광석 현미경을 수행 할 준비가 될 때까지. </li><li> 이미지 수집 및 공 초점 현미경 (실험 조건에 블라인드)와 실험의 분석. 60X 오일 NA 다음 레이저와 1.49 침수 목적 : 아르곤 가스를 488 나노 미터, 561 다이오드, 640 다이오드. <ol><li> 동일한 화상 취득 설정을 사용하여, 신경 세포의 세포체 및 포커스 몇개 수지상 프로세스와 단일 Z 단면 이미지를 얻기. </li><li> 모든 엔도 시토 시스 데이터의 분석에 대해 동일한 임계 값을 사용하여, 신경 세포 당 3 ~ 4 근위 수상 돌기의 20 μm의를 따라 α-bungarotoxin 알렉사 형광 신호 및 GFP의 면역 염색을 정량화. </li><li> 여러 독립적 인 연결을 문화와 실험을 반복, 각 시점에 10-12 뉴런에서 데이터를 분석 할 수 있습니다. </li></ol></li></ol>

<ol>
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Pharmacol. 76, 992-997 (2009).</li><li>Terunuma, 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=Prolonged+activation+of+NMDA+receptors+promotes+dephosphorylation+and+alters+postendocytic+sorting+of+GABAB+receptors.">Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 13918-13923 (2010).</li><li>Goslin, K. G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Nerve+Cells.">Culturing Nerve Cells.</a> , (1998).</li></ol>

저자가 공개하는 게 없다.

여기에 설명 BBS 기반 고정 라이브 기법 수용체 또는 세포주, 뉴런, 및 다른 프라이 머리 셀의 다른 세포막 단백질 매매를 추적하기 위해 사용될 수있다. 이 방법은 성공적 막 삽입 및 리간드 관문 이온 채널과 GPCR의 제거를 연구 인해 수용체 작용제 및 조절제의 존재로 매매의 변화를 평가하기 위해 사용되었다. 주요 측면은 적절한 세포 위치 태그의 현지화 및 BBS의 추가를 확인하기 위해 컨트롤을 ...

의 형광 수용체 현지화 및 역학을 연구하는 α-bungarotoxin을 결합 사용은 니코틴 성 아세틸 콜린 수용체 13-15, 독소의 내생 대상의 연구에서 개척되었다. 그 후, 최소한의 BGT 바인딩 펩타이드 (BBS)의 결합은 두 흥분성과 억제 리간드 게이트 이온 채널 및 G 단백질 결합 수용체 2,10,16-21의 인신 매매를 연구하는 데 사용되었습니다. 이 BBS 기반 기술은 표면의 바이오 티 닐화 방법, 세포 항원에 대한 항체와 살아있는 세포의 항체 라벨 및 (FRAP)을 photobleaching에 후 형광 복구와 같은 인신 매매의 연구에 사용되는 다른 방법에 이점을 제공한다. 세포 표면 동안 바이오 티 닐화 무료 아민이 공유 결합으로 세포 활동에 영향을 미칠 가능성과 함께 수정됩니다. 항체 기반의 연구는 종종 인신 매매 이벤트를 변경할 수있는 표면 항원의 클러스터링이나 모자를 씌우기에 의해 방해되었다. 때문에 FRAP들에 대한 표백 단계에tudies, 중요한 문제는 기본 세포 구조를 손상된다. 추가적인 장점은 BBS는 구조도 바이오틴 결합 엉덩이에 bungarotoxin 수용체 인신 매매와 생화학 적 방법에 사용할 수있는 태그입니다. 이 기술은 세포주 및 일차 전지가 쉽게 적용 가능하다. 표시된 바와 같이 니코틴 아세틸 콜린 (nAChR의) 수용체를 발현하는 세포에 사용하기위한, nAChR의 길항제 tubocurarine는 프로토콜에서 사용되어야한다. tubocurarine의 부재에 형질 감염되지 않은 세포 (엔도 시토 시스 프로토콜 T = 0 시점에 해당) 간단한 표면 BGT 라벨을 수행하면 내생 nAChR의 증거를 제공 할 것입니다. 그것은 또한 단백질이 세포막에 전달되는 세포의 위치에 존재하도록 이러한 기술을 사용하기위한 중요한 고려 사항은 BBS의 적절한 삽입이다. 예를 들어, GABAAR 서브 유닛의 N-말단 도메인은 인신 동안 기공을 갖는 루멘에 상주ficking 및 세포 표면 수용체와 세포 내 이입에 의해 이벤트가 세포 표면에서 그들의 제거의 평가의 구체적인 표시를 허용 세포막에있는 수용체 삽입 후 세포가된다. 우리는 이전에 GFP, MYC, 또는 BBS의 추가 GABAAR 서브 유닛이 도메인에 항원 것으로 나타났습니다 것은 기능적으로 침묵이다. 표준 컨트롤은 적절 지역화 것을 태그로 단백질을 구성하는 태그없는 유사한 수준으로 발현 될 수 있도록 수행되고, 그것은 수용체 기능에 영향을주지해야한다. 형질 구조의이 특성은 문제 해결의 과발현 문제에 도움이됩니다.

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			<td style="width:201px;">Company</td>
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			<td height="20" style="height:20px;width:227px;">P3 Primary Cell 4D kit&#160;</td>
			<td style="width:201px;">Lonza</td>
			<td style="width:196px;">V4XP-3024</td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 647 conjugate</td>
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			<td style="width:196px;">B35450</td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 488 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13422</td>
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			<td height="40" style="height:40px;width:227px;">&#945;-bungarotoxin, Alexa Fluor 594 conjugate</td>
			<td style="width:201px;">Molecular Probes</td>
			<td style="width:196px;">B13423</td>
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			<td height="20" style="height:20px;width:227px;">(+)-Tubocurarine chloride</td>
			<td style="width:201px;">Tocris</td>
			<td style="width:196px;">2820</td>
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			<td height="20" style="height:20px;width:227px;">mounting medium&#160;</td>
			<td>DAKO</td>
			<td style="width:196px;">cS703</td>
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			<td height="20" style="height:20px;">DPBS no calcium,magnesium&#160;</td>
			<td style="width:201px;">Invitrogen&#160;</td>
			<td style="width:196px;">14190-136</td>
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			<td height="20" style="height:20px;width:227px;">poly-D-lysine&#160;</td>
			<td style="width:201px;">Sigma</td>
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			<td height="20" style="height:20px;width:227px;">gephyrin antibody</td>
			<td>Synaptic Systems</td>
			<td>147 011</td>
			<td>1:300</td>
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			<td height="20" style="height:20px;width:227px;">VIAAT/VGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 002</td>
			<td>1:1000</td>
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			<td height="20" style="height:20px;width:227px;">anti GFP</td>
			<td style="width:201px;">Life Technologies</td>
			<td>A6455</td>
			<td>1:2000</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:227px;">glass bottom tissue culture dish</td>
			<td style="width:201px;">MatTek Corporation</td>
			<td colspan="2">P35G-1.5-14-C - Mattek Dishes</td>
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			<td height="40" style="height:40px;width:227px;">coverslips (round cover glass), #1 thickness, 12 mm</td>
			<td style="width:201px;">Warner Instruments</td>
			<td style="width:196px;">640-0702</td>
			<td></td>
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using an &#945;-bungarotoxin binding site tag to study gaba a receptor membrane localization and trafficking

그것은 점점 더 분명 그 이온 성 GABA 수용체 (GABAAR), 전시 매우 역동적 인 인신 매매와 세포 표면의 이동성 1-7 등의 신경 전달 물질 수용체,. 수용체 세포 표면의 현지화 및 엔도 사이토 시스를 연구하기 위해, 여기에 설명 된 기술은 세포가 α-bungarotoxin (BGT) 결합 부위 (BBS)를 포함하는 구조를 표현하는 형광 α-bungarotoxin의 사용을 결합한다. BBS (WRYYESSLEPYPD)는 높은 친화력 8,9와 BGT를 결합하는 근육 니코틴 성 아세틸 콜린 수용체의 α ​​서브 유닛을 기반으로합니다. BBS 사이트의 설립 이전에 GABAA 및 대사성 GABAB의 추적 2,10 수용체에 설명 된대로, 표면의 현지화 및 수용체의 삽입 또는 외인성 형광 BGT의 응용 프로그램 제거를 측정 할 수 있습니다. BBS 사이트 또, 아미노산 및 4 표준 m 의하여 성숙한 GABAAR 소단위의 (5) 사이에 pH 민감성 GFP (pHGFP 11)을 삽입olecular 생물학 및 PCR 클로닝 전략 12 (그림 1 참조). BBS는 13 아미노산 알라닌 / 프로 링커에 의해 분리 된 pH 민감성 GFP 기자의 3 &#39;입니다. 총의 기자가 BGT 정상화 총 수용체 인구 수용체 인구를 표시하므로 GABAAR 소단위 단백질 수준을 태그로 고정 샘플을 기반으로이 책에서 설명한 연구 인신 매매의 경우, pHGFP는 역할을한다. 이것은 태그 GABAAR 소단위의 높거나 낮은 기준선 식의 결과 BGT 염색 신호 가변성 세포 간을 최소화한다. 또한 pHGFP 태그 라이브 또는 고정 이미징 실험을위한 구조 발현하는 세포를 쉽게 식별 할 수 있습니다.

BBS 태그 구조의 특성이 같은 표현 단백질 (특히 여러 개의 서브 유닛으로 구성 수용체)가 제대로 조립 여부를 결정하는 중요한 컨트롤이 포함되어, 세포 표면에 교통 정체를 적절하게 지역화. 억제 시냅스는 억제 비계 단백질 gephyrin와 colocalize과 시냅스 소포 (VIAAT / VGAT)에 GABA와 글리신을로드하는 기공 억제 아미노산 수송에 의해 확인 시냅스 억제 터미널, 나란히 놓여있다 수용체 표면 클러스터는...

Watch this Scientific Journal Video about Using an Î±-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking at JoVE.com

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

여기에서 우리는 GABA 해마 신경 세포에있는 수용체 표면의 현지화 및 엔도 사이토 시스를 측정하기 위해-bungarotoxin을 α에 결합 형광 알렉사 염료의 사용을 보여줍니다. α-bungarotoxin, 세포막 단백질의 세포 내 이입 매매 분석을 달성 할 수있는 바인딩 짧은 세포 태그 베어링 구조의 사용을 통해.

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

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

using-an-bungarotoxin-binding-site-tag-to-study-gaba-receptor

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Neuroscience

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

15.7K 조회수.  University of Pittsburgh School of Medicine.  여기에서 우리는 GABA 해마 신경 세포에있는 수용체 표면의 현지화 및 엔도 사이토 시스를 측정하기 위해-bungarotoxin을 α에 결합 형광 알렉사 염료의 사용을 보여줍니다. α-bungarotoxin, 세포막 단백질의 세포 내 이입 매매 분석을 달성 할 수있는 바인딩 짧은 세포 태그 베어링 구조의 사용을 통해. 

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

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

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

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

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

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

연구

신경과학

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

공간 탐색 구역에서 &#39;학습 연구에 적외선 분광학 가까이 MazeSuite 및 기능 사용

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

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

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

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

이미징은 기본 양식 마우스 뉴런의 플라즈마 막에 수용체 삽입을 pHluorin 태그

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

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

마우스 뇌 슬라이스의 Nicotinic 아 세티 콜린 수용체 기능을 공부하는 약물의 로컬 응용 프로그램

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

막 성인 쥐에서 Ventromedial 시상 하부 뉴런의 잠재적 인 염료 이미징은 포도당 감지를 연​​구하는

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

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

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

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

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

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

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

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

RNA에 의한 Locust Antenna 내의 악취 수용체 유전자의 국산화<em&gt; 현장에서</em&gt; 하이브리드 화

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

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

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

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

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

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

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

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

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

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

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

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