대부분의 작업과 관련된 인력은 NIH 보조금 NS060677에 의해 부분적으로 NIH가 MH099559과 (BSK에) MH10406​​9 부여에 의해 지원되었다. 작품의 일부는 CHDI 재단에 의해 지원되었다.

모든 동물 프로토콜은 실험 동물의 관리 및 사용을위한 건강 가이드의 미국 국립 연구소에 따라이었고, UCLA에서 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1.1) 마이크로 피펫 및 AAV2 / 5 바이러스로드 준비 <ol><li> 바이러스의 주입을위한 좋은 붕규산 유리 마이크로 피펫을 사용합니다. 수직 풀러와 두 단계 당기는 프로그램을 사용하여 마이크로 피펫을 당깁니다. 베벨 피펫 분쇄기를 이용하여 40 °의 각도로 피펫. 40 μm의 6 생크 길이 - - 7 MM 사용하기에 이상적이다 피펫 (20)의 팁 직경을 가질 것이다. 피펫 및 수술 도구를 압력솥. 드릴을 70 % 에탄올로 닦아 소독한다. </li><li> 10 μL 씩에 -80 ° C에서 바이러스 AAV2 / 5 GfaABC 1 D.cyto-GCaMP3.SV40 (1.5 × 10 13 GC / ㎖)을 저장합니다. 그냥 수술 전, 냉동실에서 분주을 꺼내 번째를 채우기 사용까지 얼음에 저장전자 마이크로 피펫. </li><li> 바이러스가 시린지 펌프를 이용하여 함께 마이크로 피펫을 채운다. 단단히 강화 플런저 유리 주사기, 피팅 적절한 크기의 튜브 압축을 통해 튜브, 조각의 한쪽 끝을 연결합니다. 맞는 적절한 크기의 이동식 바늘 압축을 통해 튜브의 조각의 다른 쪽 끝으로 멸균 마이크로 피펫을 연결합니다. </li><li> 바이러스 벡터를 넣기 전에, 바늘 1ml의 주사기 수단 적색 IV (~ 1 ㎎ / 50ml)로 컬러 미네랄 오일 시스템 (27 G를 충전함으로써 튜브, 주사기 및 마이크로 피펫을 포함한 배기계로부터 공기 방울을 제거 ). </li><li> 유리 마이크로 피펫에서 파라 필름의 깨끗한 부분을 놓고 5 분배 - 파라 필름에 바이러스 벡터의 10 μl를. </li><li> 역방향 0.5 μL / 분으로 주사기의 플런저를 이동시킴으로써 바이러스 벡터를 빨아 및 유리 피펫에 벡터 및 오일 사이의 경계를 표시한다. </li></ol> 1.2) 마취, 머리 자르기, H개두술에 대한 EAD 고정 및 준비 <ol><li> N 2 O 및 O 2, 5 % 이소 플루 란 채워진 챔버 내로 마우스 (P49-63, C57BL / 6)을 넣어. 목적이있는 운동의 손실 둔화 호흡 (- 90 / 분 ~ 60)에 의해 마취의 깊이를 평가한다. 마우스를 마취 할 때 머리에 머리를 잘라. </li><li> 머리가 마취 및 환기 시스템에 배치 무딘 귀 바, 코에 의해 확보와 함께, 정위 프레임에 마우스를 맞 춥니 다. 2-3%에서 연속 이소 플루 란을 유지한다. 마우스가 마취하에 자신의 깜빡임을 잃게하기 때문에 두 눈에 인공 눈물 연고를 적용합니다. </li><li> 통증 완화를위한 피하 주사에 의해 프레 놀핀 (0.1 ㎎ / ㎖)의 0.05 mL로 관리 할 수​​ 있습니다. </li><li> 10 % 포비돈 요오드 및 70 % 알코올로 3 회 면도 외과 영역, 두 용액 사이에서 교호 포비돈 요오드로 시작. 전면에서 시작하고 이미 잘라 머리카락을 제거하기 위해 뒤쪽으로 닦습니다. </li><li> 상단 O에 피부 절개를눈 사이에 시작하고 귀 사이에 끝나는 머리 F. </li></ol> 1.3) 개두술 Microinjections <ol><li> 면봉으로 쓸어 넘겨 골막을 분리 한 다음 치과 면봉과 두개골의 표면을 건조. 주사 부위 주위에 마크를 만들고, 고속 드릴에 의해 구동 소형 강철 버어를 사용하여 마크의 에지 주위 드릴. </li><li> 뼈를 제거하고 식염수로 표면을 청소합니다. </li><li> 전후방 0.8, 내측 - 외측 : 다음 좌표 (mm)과 선조체 (striatum) 측면 왼쪽 지느러미에 피펫을 놓고 +2.0, 지느러미 - 복부를 pial 표면 : -2.4 (13). 이 좌표는 미세 주사 바늘의 끝이 약간이 핵 내에 침투 즉, 단지 배 외측 선조체 (그림 1A, B) 위에 앉아 있다는 것을 의미합니다. 손상 혈관을 피하십시오. 피펫은 혈관에 바로 될 일이있는 경우, 버스를 조정0.2 mm - 0.1 ~에 의해 조정합니다. </li><li> 0.2 μL / 분으로 설정 주입 속도로 주입을 시작하고 일반적으로 바이러스의 1-1.5 μl를 주입. </li><li> 10 분 동안 주입 한 후 장소에 피펫을두고 천천히 피펫을 철회. </li><li> 연속 봉합 외부 나일론 봉합 수술 상처를 닫아 마칩니다. </li></ol> 1.4) 복구 <ol><li> 24 시간 동안 가열 패드에 깨끗한 케이지에 마우스를 넣습니다. 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 마우스를 반환하지 않습니다. 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 마우스를 방치하지 마십시오. </li><li> 트리 메소 프림 Sulfamethoxiazole 추가 일주일 동안 물 (40 mg을 트리 메소 프림과 200 mg을 sulfamethoxiazole이 포함 된 500 ml의 물에 5 ml의). 수술 후 프레 놀핀에게 최대 3 일 동안 하루에 두 번 관리합니다. </li><li> , 먹이 12 시간 명암주기에 마우스를 유지하고 매일 물을차 수술 후 3 일에 한 번 일까지 당 가중된다. 이 3 일 동안, 10 % 이상의 체중 감량과 함께 임의의 마우스는 손실, 대신 실험 실시되는 안락사 될 것으로 간주된다. </li><li> 별도로, 일주일에 두 번 마우스 케이지를 변경하고, 적어도 하루에 한 번 건강을 모니터링. 정위 microinjections 후 3 주 - 일반적으로 마우스는 2 내에서 사용된다. </li></ol> 공 초점 칼슘 이미징 2. 급성 뇌 조각 준비 절단 및 녹화 솔루션 2.1) 준비. <ol><li> (MM)을 포함하는 용액을 절단 준비 : 194 크로즈, 30의 NaCl, 4.5 KCl을 10 D 글루코스, 하나의 MgCl 2, 1.2의 NaH 2 PO 4, 95 % O 2 및 5 % CO 2로 포화 된 26의 NaHCO3를. </li><li> 포함하는 기록 용액 (mM)을 준비한다 (124)의 NaCl, 4.5 KCl을, 하나의 MgCl 2, D-10 글루코스,이 CaCl2를 1.2의 NaH 2 PO 4 </서브&gt;, 26의 NaHCO3; pH가 7.3-7.4을, 290 - 95 % O 2 및 5 % CO 2로 포화 295 mOsm. 레코딩 솔루션과 뇌 조각을 들고 비커를 입력하고 34 ℃에서 보관하십시오. </li></ol> 2.2) 슬라이스 <ol><li> 2-3 주 정위 microinjections 후, 깊이와 끝으로 마우스를 마취 한 다음 목을 벨. </li><li> 뇌를 추출하고, uninjected 우반구를 제거하는 블레이드를 사용하고, 강력 접착제를 사용하여 vibratome 트레이에 왼쪽을 탑재합니다. 차가운 얼음 절단 버퍼와 vibratome 트레이를 입력하고 95 % O 2 5 % CO 2로 포화 유지. </li><li> 300 μm의 두께로 관상 또는 시상 선조체의 뇌 조각을 잘라. 일반적으로, 4-5 관상, 또는 6-7 시상 선조체 슬라이스는 수집 할 수 있습니다. </li><li> 34 ° C에서 따뜻하게 슬라이스 잡고 비커에 조각을 이동하고, 이후 recordi 실온에서 그들을 저장하기 전에 30 분 동안 그들을 거기 유지NG. </li><li> [칼슘] 난 촬상 전에 적어도 30 분 동안 실온에서 산화 된 기록 버퍼에서 뇌 조각 부화. </li></ol> 3. 면역 조직 화학 (IHC) <ol><li> 두 주 바이러스 주사 후, PBS로 transcardially 가진 마우스 PBS에서 10 % 포르말린 관류 하였다. </li><li> 제거 하룻밤 후 수정, 적어도 2 일 동안 버퍼링 30 % 자당의 뇌를 cryoprotect. </li><li> 그라 이오 스탯 마이크로톰을 사용하여 40 μm의 섹션을 준비합니다. </li><li> 10 분 간격으로 섹션을 PBS로 3 회 헹군다. </li><li> 10 % 정상 염소 혈청과 PBS에서 1 시간 동안 0.5 % 트리톤 X-100으로 섹션을 품어. </li><li> 0.5 % 트리톤 X-100와 PBS에서 준비 차 항체로 4 ℃에서 하룻밤 섹션을 품어. 다음과 같이 사용되는 일차 항체이었다 : 닭 항 GFP 1 : 1,000, 마우스 항 S100β 1 : 400, 마우스 항 NeuN 1 : 600, 마우스 항 글루타민 신테 1 : 300. </li><li> 10m에서 섹션을 PBS로 3 회 씻어간격. </li><li> 실온에서 2 시간 동안 10 % 정상 염소 혈청과 함께 PBS에서 제조 된 이차 항체 섹션을 인큐베이션. 다음 두 번째 항체는 같았다 : 염소 항 - 마우스 Alexa546 1 : 500, 염소 항 - 닭 Alexa488 1 : 500. </li><li> 천천히 유리 커버 슬립과 섹션을 포함, 건조 유리 슬라이드에 섹션을 마운트하고 antifade 설치 매체의 몇 방울을 적용합니다. 4 ° C에서 슬라이드를 저장합니다. </li><li> 1.3의 개구와 40X 기름 침지 렌즈와 공 초점 현미경에서 이미지를 가져 가라. </li></ol> 4. 공 초점 [칼슘 2 +] 나는 영상 참고 : [칼슘 2 +] 나는 0.8의 개구와 40 배 침수 대물 렌즈와 공 초점 현미경을 사용하여 수행되었다 이미징. <ol><li> 1 ㎖ 당 2 - 시간 1 ~ 5의 기록 챔버 내에서, 도시 슬라이스 및 슬라이스 (1)의 유량을 갖는 실온에서 산소 기록 버퍼와 superfuse 후분. 그 후 실험 기간 동안 움직임을 최소화하기 위해 슬라이스 위에 나일론 문자열 백금 하프 배치. </li><li> 시각화 및 10 배 침수 목적에서 밝은 필드 발광과 등쪽 선조체 (striatum)를 찾습니다. </li><li> 40 배 침수 대물 렌즈로 전환 한 아르곤 레이저의 488 nm의 라인을 켭니다. 초점면을 변경, 소마, 지점과 branchlets의 기초 형광 표시, 약 20 μm의 슬라이스 표면 아래에있는 세포를 찾을 수 있습니다. 참고로, 손상된 세포는 일반적으로 피해야한다 평균 이상 형광 수준을 표시합니다. </li><li> 스캔을 시작하는 &#39;프레임 스캔&#39;모드를 사용합니다. 통상적으로, 0.5로 조정 한 레이저 강도 - 최대 출력의 5 % [칼슘] 전 사이토-GCaMP3 가진 이미지에 충분하다. 성상 세포의 전체 영역에 [칼슘 2 +] 나는 역학을 모니터링하기위한 프레임 이상의 속도 당 1 초의 스캔 속도와 &#39;프레임 스캔&#39;추천!입니다nded하지만,이 문제의 실험에 따라 결정해야합니다. </li><li> 보기의 전체 필드 2 성상 세포 - 1을 모니터 3 배 - 필요한 경우, 이미지에 [칼슘 2 +] 나는 과정에서, 2의 디지털 배율을 사용합니다. </li><li> 녹화 중에 포화 픽셀을 방지하기 위해, 세포를 사색하는 &#39;히로&#39;조회 모드를 사용합니다. 항상 붉은 색은 채도의 표시로 나타나는 경우 테스트하기 위해, 약 5 분 동안 &quot;파일럿 녹화&quot;로 시작합니다. 이 경우, 레이저 출력을 낮추거나 PMT를 낮추고 / 이득 / 오프셋 값과 화소 값은 비 포화 영역에있는 테스트하는 다른 파일럿 기록을 수행한다. - 5 % (10 메가 와트), PMT 550-650 볼트, 게인 2.0 - 레이저 출력 전력 0.5 3.5 배, 0 오프셋 : 일반적으로 결합 된 매개 변수 영상 성상 세포에 사용 [칼슘] 선조체에서 나는 다음과 같습니다 - 5 %. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+near+plasma+membrane+and+global+intracellular+calcium+dynamics+in+astrocytes.">Measuring near plasma membrane and global intracellular calcium dynamics in astrocytes.</a> J Vis Exp. 26, (2009).</li><li>Reeves, A. M., Shigetomi, E., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+loading+of+calcium+indicator+dyes+to+study+astrocyte+physiology:+key+limitations+and+improvements+using+morphological+maps.">Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps.</a> J Neurosci. 31, 9353-9358 (2011).</li><li>Li, D. D., Agulhon, C., Schmidt, E., Oheim, M., Ropert, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+investigating+astrocyte-to-neuron+communication.">New tools for investigating astrocyte-to-neuron communication.</a> Frontiers in Cellular Neuroscience. 7, (2013).</li><li>Davila, D., Thibault, K., Fiacco, T. A., Agulhon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+molecular+approaches+to+understanding+astrocyte+function+in+vivo.">Recent molecular approaches to understanding astrocyte function in vivo.</a> Front Cell Neurosci. 7, 272 (2013).</li><li>Paxinos, G., Franklin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2012).</li><li>Perea, G., Yang, A., Boyden, E. S., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+astrocyte+activation+modulates+response+selectivity+of+visual+cortex+neurons+in+vivo.">Optogenetic astrocyte activation modulates response selectivity of visual cortex neurons in vivo.</a> Nat Commun. 5, 3262 (2014).</li><li>Sofroniew, M. V., Vinters, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes:+biology+and+pathology.">Astrocytes: biology and pathology.</a> Acta Neuropathol. 119, 7-35 (2010).</li><li>Eid, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+glutamine+synthetase+in+the+human+epileptogenic+hippocampus:+possible+mechanism+for+raised+extracellular+glutamate+in+mesial+temporal+lobe+epilepsy.">Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy.</a> Lancet. 363, 28-37 (2004).</li><li>Eid, T., Williamson, A., Lee, T. S., Petroff, O. A., de Lanerolle, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+astrocytes--key+players+in+human+mesial+temporal+lobe+epilepsy.">Glutamate and astrocytes--key players in human mesial temporal lobe epilepsy.</a> Epilepsia. 49 Suppl 2, 42-52 (2008).</li><li>Tong, X., 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=Astrocyte+Kir4.1+ion+channel+deficits+contribute+to+neuronal+dysfunction+in+Huntington's+disease+model+mice.">Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice.</a> Nat Neurosci. 17, 694-703 (2014).</li><li>Ortinski, P. 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=Selective+induction+of+astrocytic+gliosis+generates+deficits+in+neuronal+inhibition.">Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.</a> Nat Neurosci. 13, 584-591 (2010).</li><li>Zhang, Y., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+heterogeneity:+an+underappreciated+topic+in+neurobiology.">Astrocyte heterogeneity: an underappreciated topic in neurobiology.</a> Curr Opin Neurobiol. 20, 588-594 (2010).</li><li>Reimsnider, S., Manfredsson, F. P., Muzyczka, N., Mandel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+transgene+expression+after+intrastriatal+pseudotyped+rAAV2/1,+rAAV2/2,+rAAV2/5,+and+rAAV2/8+transduction+in+the+rat.">Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat.</a> Mol Ther. 15, 1504-1511 (2007).</li></ol>

저자가 공개하는 게 없다.

여기에 설명 된 방법은 우리가 현장에서 후속 [칼슘 2 +] 나는 영상을 위해 생체 내에서 선조체의 성상 세포에서 사이토-GCaMP3을 표현 할 수있다. 이 방법은 형질 전환 또는 강력한 대상 단백질, 신속성의 표현과 실험적인 구현 및 해부학 적 특이성의 유연성을 포함 노크에서 마우스를 사용하여보다 이점이있다. AAV2 / 5 사용 GCaMP3의 발현은 구체적이고 강력한 것으로 밝?...

성상 세포는 뇌의 유비쿼터스와 풍부한 신경 교세포 있습니다. 그것은 잘 성상 세포가 중요한 지원 및 세포 외 공간에서의 K + 농도의 버퍼링, 흡수 신경 전달 물질의를 제공 할뿐만 아니라 영양소를 포함 항상성 역할을 역할을한다는 설정됩니다. 그러나, 최근의 연구들은 또한 자연적으로 발생하여 신경 세포의 활동을 1 증가 [칼슘] 내가 신호를 표시하는 것을 알 수있다. 성상 세포의 존재는 [칼슘 2+] I 시그널링 점점 뉴런과의 통신을 유발하는 것으로 생각되고 있으며, 이와 같이 성상 내의 &quot;칼슘 흥분성&quot;의 형태로 해석되었다. 지난 20 년 동안 사용할 수있는 데이터는 성상 세포와 신경 세포는 아마도 양방향 방식으로 통신 할 수있는 두 가지 설정을 제안한다. 첫째, 성상 세포는 종종 내가 신경 전달 물질에 의해 활성화 [칼슘]의 증가로 응답신경이 해제 신경 조절. 둘째, [칼슘 2 +] 성상 세포 내에서 내가 증가는 다시 신경과 혈관에 영향을 미칠 수있는 성상 세포에서 신호 전달 분자의 방출을 야기한다. 증거는 성상 세포에서 방출 분자가 성상 세포 간 신경 신호를 통해 ​​시냅스 회로와 궁극적으로 행동 3-5의 기능의 변화로 이어질 것이 좋습니다. 그러나이 급속히 발전 연구 분야 남아 있고, 이는 성상 세포의 더 상세한 이해는 [칼슘 2+] I는 현재의 불확실성 (6)의 일부를 해결하기 위해 필요하다고 주장하고있다. 과거의 연구에서,이 성상 세포에 유기 칼슘 지표 염료의 대량로드가 확실하게 감지하지 못하는 것을 증명했다 [칼슘 2 +] 문화 및 현장 7-10에서 전체 성상 세포 내에서 내가 신호. 이러한 연구 결과는 우리와 다른 사람 6,11,12에 의해 논의되었다. emerginG 사진은 [칼슘 2 +] 나는 신경과 혈관과의 상호 작용에 대한 기본 사이트입니다 성상 세포 프로세스 (예를 들면, 지점 및 branchlets) 내에서 신호를 거의 자세히 살펴되어 있지 않은 것입니다. 최근에는 세포질 GCaMP3, GCaMP5G 및 GCaMP6 및 세포막과 같은 유전자 인코딩 칼슘 지표 (GECIs)의 사용이 닿는 버전 (예를 들어, Lck에-GCaMP3)는 성상 세포 등의 작은 구획에 [칼슘] 내가 신호의 연구를 허용하고있다 얇은 프로세스, 세포막 근처 전체 지역 7,8 내. 그러나 GECIs 유기 칼슘 2 + 지표 염료를 통해 한 가지 단점을 가지고 GECIs 적절하게 할 표현하는 유전 적 방법은 주 단위로 생체 내에서 성상 세포에 선택적으로 인코딩 유전자를 전달하기 위해 그 요구 사항입니다. 생체 내에서 발현은 일반적으로 노크에 쥐 또는 바이러스를 기반으로 배달 응용 프로그램과 함께, 형질 전환 마우스를 사용하여 달성된다바퀴벌레. 본 조브 기사에서 우리는 아데노 - 관련 바이러스를 사용하여 선조체 성상 세포에 GECIs을 제공하기 위해 사용되는 방법과 절차를보고합니다. 우리는 예를 들어 사이토-GCaMP3에 초점을 동일한 기본 절차는 다른 GECI 또는 형광 단백질 기반 기자 모든 작동합니다.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>704506</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Capillaries</td>
			<td >World Precision Instruments</td>
			<td >1B100-4</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Narishige</td>
			<td >PC-10</td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipette grinder</td>
			<td >Narishige</td>
			<td >EG-40</td>
			<td></td>
		</tr>
		<tr >
			<td >pZac2.1 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I mL syringe</td>
			<td >BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr >
			<td >syringe needle</td>
			<td >BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr >
			<td >AAV2/5 virus</td>
			<td >UPenn vector core</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan red IV</td>
			<td >Sigma-Aldrich</td>
			<td >67386</td>
			<td></td>
		</tr>
		<tr >
			<td >Mineral oil</td>
			<td >CVS Pharmacy</td>
			<td>152355</td>
			<td></td>
		</tr>
		<tr >
			<td >Cryostat</td>
			<td >Leica</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
			<td >900LS</td>
			<td></td>
		</tr>
		<tr >
			<td >High Speed Rotary Micromotor Kit</td>
			<td >FOREDOM</td>
			<td >K.1070</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Santa cruz biotechnology</td>
			<td >sc-281692</td>
			<td></td>
		</tr>
		<tr >
			<td >Super Glue</td>
			<td>Krazy&#174;Glue</td>
			<td>KG925</td>
			<td></td>
		</tr>
		<tr >
			<td >Microslicer</td>
			<td >Ted Pella</td>
			<td >DTK-Zero 1</td>
			<td></td>
		</tr>
		<tr >
			<td >Confocal microscopes</td>
			<td >Olympus</td>
			<td >FV300 and FV1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Normal goat serum</td>
			<td >Vector</td>
			<td >S-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >chicken anti-GFP</td>
			<td >Abcam</td>
			<td >ab13970</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-s100&#946;</td>
			<td >Sigma-Aldrich</td>
			<td >S2532</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-NeuN</td>
			<td >Millipore</td>
			<td >MAB377</td>
			<td></td>
		</tr>
		<tr >
			<td >mouse anti-glutamine synthetase</td>
			<td >Millipore</td>
			<td >MAB302</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-mouse-Alexa546</td>
			<td >Invitrogen</td>
			<td >A11003</td>
			<td></td>
		</tr>
		<tr >
			<td >goat anti-chicken-Alexa488</td>
			<td >Invitrogen</td>
			<td >A11039</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover Glass</td>
			<td >Fisher Scientific</td>
			<td >12-548-5J</td>
			<td></td>
		</tr>
		<tr >
			<td >Mounting Medium</td>
			<td >Vector</td>
			<td >H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging intracellular ca2+ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

줄무늬 체에서 사이토-GCaMP3의 성상 세포 특이 적 발현을 위해, 우리는 이전에 견고 GCaMP3 및 리포터 유전자를 구동하는 도시 된 아데노 - 관련 바이러스 (AAV) 5 혈청 형 및 GFAP GfaABC 한 D 촉진제 (도 1A)를 사용 해마와 대뇌 피질의 성상 세포 8, 14의 식입니다. 2 주 마우스 선조체에 바이러스 미세 주입 한 후, 마우스 (~ 10 주)을 관류하고 IHC는 선조체 (그림 1B)의 ?...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

이미징 세포 내 칼슘<sup&gt; 2 +</sup&gt; 유전자 인코딩 된 칼슘 표시기를 사용하여 성인 마우스의 선조체 성상의 신호

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

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JoVE Journal

Neuroscience

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

14.7K 조회수.  University of California Los Angeles. The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ. 

비디오: Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

영상의 연결 응답 Vomeronasal 조직의 슬라이스 준비에 유전적으로 인코딩된 칼슘 센서를 표현

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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신경과학

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

확인된 뉴런과 먹이 행동에서 칼슘 신호의 동시 녹화 Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

모델링 성상 세포종 병인<em&gt; 체외</em&gt;와<em&gt; 생체</em&gt; 대뇌 피질의 성상이나 조건, 유전자 조작 쥐에서 신경 줄기 세포를 사용하여

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

유전자 인코딩 된 형광 전압 센서의 두 가지 유형의 잠재적 이미징 막

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

뉴런의 유전자 인코딩 된 부 자연스러운 아미노산을 사용하여 신경 세포 단백질의 광 제어

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

로컬 Ca2 + 신호 막 대상 Ca2 + 지표에 의해 경작된 한 세포에의 해 부

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

정상 및 헌팅턴 마우스의 급성 뇌 슬라이스에서 글루타메이트 방출 및 섭취량의 단일 시냅스 지표

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

성상 세포에서 세포기관 및 기타 화물의 세포내 운송 시각화 및 분석

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

성인과 노화 마우스에서 해마 조각에서 저산소증 최소화

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.