This work was partially supported by National Institute of Deafness and Other Communications Disorders Awards R03-DC-012125 to D. A. Llano and F31-DC-013501 to B. J. Slater as well as the Carver Foundation.
The authors would like to thank Jason MacLean and Matthew Banks for technical advice with calcium imaging.

모든 절차는 일리노이 대학에서 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 모든 동물 실험 동물 관리의 인증에 대한 미국 협회의 승인을 동물 보호 시설에 수용되었다. 모든 시도가 사용 동물의 수를 최소화하고 연구의 모든 단계에서의 고통을 감소시키기 위해 만들어졌다. 및 공격 태도를 보여준 마우스에서 뇌의 제거 1. 준비 <ol><li> 관류 및 슬라이스 배양을 위해 준비합니다. <ol><li> 약 30 분 슬라이스 전에 녹음 또는 영상 이전 슬라이스의 배양을위한 높은 크로스 커팅 솔루션 및 저 칼슘 ACSF을 준비합니다. </li><li> 모든 필요한 도구 (대형 무딘 끝 가위, 작은 봄 가위, 해부 포셉, 큰 가위 단두대, 홍채 가위, 보석상의 집게, 27G X 1/2 인치 바늘로 10 ML의 주사기, 1cm X 2cm 필터의 작은 조각을 배치합니다 종이 및 SL에 대한 깨진 팁 파스퇴르 피펫관류 및 슬라이싱 및 관류 트레이를 준비 얼음 전송). </li><li> 32 O C의 물을 욕조와 솔루션을 절단 가득 배양 접시에 산소 ACSF와 실을 배양 설정합니다. </li></ol></li><li> 공격 태도를 보여준 무대 절단 준비합니다. <ol><li> 약 1cm 3 범프의 뇌에 대한 역전과 1.5 cm X 1.5 mm X 3mm로 사용할 3 % 한천의 작은 조각을 잘라하는 IC를 지탱하는 데 사용합니다. </li><li> 시아 노 아크릴 레이트 접착제 스테이지뿐만 아니라 그들이 80 O 각도를 형성하도록 역전의 오른쪽에 범프 상 역전을 붙인다. </li></ol></li><li> 뇌를 제거합니다. <ol><li> 깊이 P12-P20 케타민 100 ㎎ / ㎏과 xylaxine 3 ㎎ / ㎏ (또는 유사한 윤리위원회의 승인 절차)와 (이상적 p14-18) 마우스를 마취. 참고 : 발가락 핀치에 응답의 부족을 통해 적절한 마취 수준을 확인합니다. 동물은 마취하에 간단히 바와 같이, 안연고는 불필요하다. </li><li> 무딘 사용가위를 종료 목에 칼 모양의 과정에서 갈비뼈를 노출. <ol><li> 칼 모양의 과정을 찾아 약 1.5 cm의 수평 절단을합니다. </li><li> 어깨에 이전 수평 절단의 끝에서 두 개의 수직 절단, 약 1.5 cm 각을 확인합니다. </li></ol></li><li> 다이아 프램 및 늑연골 접합을 극복하는 것은 마음을 노출. </li><li> 작은 스프링 가위, 마음의면을 지느러미에 복부에서 우심방으로 잘라 작은, 2-5mm합니다. </li><li> 1/2 인치 X 27G 바늘 10 ㎖ 주사기를 사용하여 좌심실을 주입하고 신속하게 높은 절삭 수크로오스 용액을 관류 동물. </li><li> 피가 명확한 실행하면, 머리를 제거하는 큰 가위를 사용합니다. 참고 : 우리는 체계적으로 관류의 유용성을 평가하지 않았습니다. 그러나, 우리의 경험에서,이 젊은 쥐 (rongeur)로 거의 100 % 성공을 함께 할 수 있고, 빨간색 (BL)을 제거하는 이점이 있는가형광 이미징 방해 할 수있는 짐 우드 세포. </li><li> 두개골을 노출하는 중간 선을 피부를 잘라. </li><li> 구부러진 홍채 가위를 사용하여 한 눈 사이의 절단, 조심스럽게 아래 뇌 손상되지 않도록 중간 선 봉합 돌보는 따라 전치부에서 잘라부터 눈 사이 두개골을 잘라. 참고 : 두라는 일반적으로 두개골과 떨어져 온다. 필요한 경우, 신중하게 뇌를 제거하기 전에 경질을 제거합니다. </li><li> 보석상의 집게 사용 놀리려는 두개골을 열고 조심스럽게 뇌를 제거하고 솔루션을 절단의 뇌를 놓습니다. 피질이 손상되지 않도록주의하십시오. </li></ol></li></ol> 2. 공격 태도를 보여준 뇌를 준비 <ol><li> vibratome 무대에 장착 뇌를 준비합니다. <ol><li> 90 O에서 두 개의 라인으로 표시된 슬라이드 오른쪽 아래 왼쪽 상단에서 17 O에서 대각선을 사용하여, 두 선이 만나는 곳, 사용, 최대 뇌 등의 측면을 배치 교차면도날은, 평평한 표면을 생성 주동이의 끝 뇌의 2-4mm를 제거합니다. </li><li> 새로 만든 평평한 표면에 뇌 꼬리 쪽을 놓고 수평 및 수직 라인과 뇌의 중간 선으로 뇌의 등쪽 표면을 맞 춥니 다. </li><li> , 17 O 라인으로 면도날을 맞 춥니 다 (30) O 각도에서 면도칼을 기울이고 이중 대각선 컷 (관상에서 17 수평면에서 O와 60 O)의 오른쪽 피질의 약 3mm를 제거합니다. </li></ol></li><li> vibratome 무대에서 뇌를 장착. <ol><li> 긴 치수는 중심선에 수직이되도록 여과지 뇌의 복부 측 1cm X 2cm의 작은 조각을 배치했다. </li><li> 신중 백스톱 (및 범프의 좌측으로) 앞의 영역에 시아 노 아크릴 레이트 접착제를 소량 적용. </li><li> 뇌와 후뇌의 꼬리 부분이되도록 홍보 접착제에에 두 번 대각선으로 잘라 뇌 얼굴을 배치범프에 opped 및 뇌의 우측에 대해 역전된다. </li><li> 섬세 전체면이 슬라이싱 챔버와 접촉하는 것을 보장하는, 뇌에 눌러. </li></ol></li></ol> 3. Colliculo-thalamocortical 슬라이스를 구하기 <ol><li> 신속하게 vibratome에서 절단 단계 및 발생하는 솔루션을 절단으로 무대를 채운다. </li><li> 뇌의 상단 (복부 측면)과 날을 맞 춥니 다. </li><li> 뇌의 상부로부터 1.5 mm를 제거 300-500 μm의 조각을 제거하고, 제거 후에 깊이를 평가한다. 참고 : IC, MGB 및 측면 geniculate 핵 (LGN)이 모두 볼 수 있습니다, 그리고 치아 이랑 (dentate gyrus)가 &#39;C&#39;모양이 600 μm의 슬라이스 한을 형성합니다. 도 3a를 참조하십시오. </li><li> 지주 (32 O를 C) 가열 된 챔버에 넣어 조각. </li></ol> 슬라이스 4. 영상 <ol><li> flavoprotein의자가 형광 이미징을위한 준비. <ol> &lt;리&gt; 표준 전기 생리 녹화 실에서 조각의 관류 ACSF을 준비, 95 %, O2 / 5 % CO 2와 버블 ACSF. </li><li> 자극 용 유리 전극을 당기십시오. 그 여러 가지 자극 전극 및 구성 (유리, 텅스텐, 탄소 섬유, 모노 폴라, 바이폴라) flavoprotein의자가 형광 이미징 모든 작업이 매우 잘 결합을합니다. </li><li> 컴퓨터, 카메라, 미세 조작기, 광원, 자극 소프트웨어, 이미지 캡처 소프트웨어를 켭니다. </li><li> 챔버에서 슬라이스를 상승 사용자 정의 다리 (보충 자료에서 사용할 수있는 디자인)를 배치, 산소 ACSF로 기록 챔버를 관류. 참고 : 관류 속도가 설정 개인에 따라 달라질 수 있습니다 5-15 ml의 / 분 여기에 사용됩니다. </li><li> 남북의 경로를 통해 하프 문자열을 배치하지 않도록주의하면서 조각을 통해 특별히 제작 된 하프를 배치하는 동안 다리를 들어 올려에서 슬라이스를 방지하기 위해 챔버 내의 액체 레벨을 낮추고, 챔버에서 슬라이스를 배치EST (그림 1A). 슬라이스 위와 아래 ACSF 흐름을 유지하기 위해 슬라이스 위의 약 1-2mm에 액체 수준을 높입니다. 주 : 재배치가 필요한 경우, 파손 단부 파스퇴르 피펫을 사용하거나 ACSF 레벨이 상승하고는 조심스럽게 집게를 사용하도록 허용. </li><li> (그림 1A 참조), ACSF 가득 유리 전극에 염화은 전극을 삽입 미세 조작기에 유리 전극을 삽입하고 아이솔레이터를 자극에 연결하고 조심스럽게 시상으로 이어지는 IC / 백질에 유리 전극을 배치합니다. </li></ol></li><li> 자가 형광 이미지 획득을 Flavoprotein. <ol><li> (무한 보정 2X 매크로 목표를 사용하여 (NA 0.13)) 4 Hz에서 이미지와 105 초 동안 카메라를 전기적으로 자극하면서 (각 자극이 0.05 Hz에서 40 Hz에서 2 밀리 초 펄스 조직의 1 초를 다섯 번으로 구성) 조명하면서 수집 푸른 빛 470-490 nm에서 515 nm의 위의 캡처와 조직. 이미지가 어느 쪽인지 확인O 어두운,도 터져 참조 그림 3 참조 열을 떠났다. 주 : 수집 시간, 컬렉션 주파수 및 자극 주파수는 실험과 같은 변형 될 수 부원료 포함 지정 프로그램에 적합하도록 변경 될 수있다. </li><li> matlab에, 및 보충 자료에서 사용할 수있는 사용자 작성 프로그램을 사용하여 내보내기 이미지는 0.05 Hz에서 이미지의 스펙트럼 전력을 분석합니다. 결과 이미지는 연결 경로를 표시합니다. 주 : 지정 프로그램은 화상을 생성하는 고속 푸리에 시계열의 화소 값의 변환을 이용한다. </li></ol></li><li> 칼슘 이미징을위한 준비. <ol><li> ACSF는 약 32 O C.을 온도를 유지하도록 가열 패드 상부 및 슬라이스의 아래쪽, 및 장소 모두에 도달 할 수 있도록 3cm 배양 접시에 올려 배양 막을 이용한 작은 배양 챔버를 준비 </li><li> 95 % O 따뜻한 ACSF 천천히 거품 작은 배양 챔버를 입력5분의 2 %의 CO 2. </li><li> 2 플루로 닉 F-127 산의 μL 및 DMSO 48 μL에 용해의 Fura - 오전 2시의 50 μg의 혼합. </li><li> 제기 막 조심스럽게 마이크로 피펫을 사용하여 작은 배양 챔버에 넣어 슬라이스 염색 직접 MGB 상기 혼합물 (또는 관심의 다른 구조)를 추가합니다. </li><li> 1 시간 - 이전 영상에 표백을 방지하고, 조각이 45 분 동안 배양 할 수 있도록 슬라이스를 커버. </li><li> 를 초과하는 물질을 씻어 10 ~ 15 분 동안 가열 보유 챔버 조각을 제거합니다. </li><li> 자극 준비와 이미지 colliculo-thalamocortical 슬라이스 4.1.6에 단계 4.1.1을 따르십시오. </li></ol></li><li> 칼슘 화상 취득. <ol><li> 365 nm의 빛과 조직을 조명 510 nm의 위에 형광을 캡처하는 동안 전기 0.2 Hz에서 (각 자극 한 2 밀리 초 펄스로 구성)를 다섯 번 조직을 자극하면서 25 초 동안 (20X 물 침지 목표를 사용) 10 Hz에서 이미지를 수집합니다. </lI&gt; <li> MATLAB과 보충 자료에서 사용할 수있는 사용자 작성 프로그램을 사용하여 내보내기 이미지는 0.2 Hz에서 이미지의 스펙트럼 전력을 분석합니다. 결과 이미지는 활성 셀을 보여줍니다. </li></ol></li></ol>

<ol>
	<li>Llano, D. A., Slater, B. J., Lesicko, A. M., Stebbings, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+auditory+colliculothalamocortical+brain+slice+preparation+in+mouse.">An auditory colliculothalamocortical brain slice preparation in mouse.</a> J. Neurosci. 111, 197-207 (2014).</li><li>Agmon, A., Connors, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamocortical+responses+of+mouse+somatosensory+(barrel)+cortex+in+vitro.">Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro.</a> Neuro. 41, 365-379 (1991).</li><li>Cruikshank, S. J., Rose, H. J., Metherate, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+Thalamocortical+Synaptic+Transmission+In+Vitro.">Auditory Thalamocortical Synaptic Transmission In Vitro.</a> J Neurophysiol. 87, 361-384 (2002).</li><li>Lee, C. C., Sherman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+and+physiology+of+ascending+streams+in+the+auditory+tectothalamic+pathway.">Topography and physiology of ascending streams in the auditory tectothalamic pathway.</a> PNAS. 107, 372-377 (2010).</li><li>MacLean, J. N., Fenstermaker, V., Watson, B. O., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+visual+thalamocortical+slice.">A visual thalamocortical slice.</a> Nat meth. 3, 129-134 (2006).</li><li>Crabtree, J. W., Isaac, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+intrathalamic+pathways+allowing+modality-related+and+cross-modality+switching+in+the+dorsal+thalamus.">New intrathalamic pathways allowing modality-related and cross-modality switching in the dorsal thalamus.</a> J. Neurosci. 22, 8754-8761 (2002).</li><li>McAlonan, K., Cavanaugh, J., Wurtz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Modulation+of+Thalamic+Reticular+Neurons.">Attentional Modulation of Thalamic Reticular Neurons.</a> J. Neurosci. 26, 4444-4450 (2006).</li><li>Weese, G. D., Phillips, J. M., Brown, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attentional+Orienting+Is+Impaired+by+Unilateral+Lesions+of+the+Thalamic+Reticular+Nucleus+in+the+Rat.">Attentional Orienting Is Impaired by Unilateral Lesions of the Thalamic Reticular Nucleus in the Rat.</a> J. Neurosci. 19, 10135-10139 (1999).</li><li>Zikopoulos, B., Barbas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+Projections+to+the+Thalamic+Reticular+Nucleus+form+a+Unique+Circuit+for+Attentional+Mechanisms.">Prefrontal Projections to the Thalamic Reticular Nucleus form a Unique Circuit for Attentional Mechanisms.</a> J. Neurosci. 26, 7348-7361 (2006).</li><li>Kalatsky, V. A., Stryker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Paradigm+for+Optical+Imaging:+Temporally+Encoded+Maps+of+Intrinsic+Signal.">New Paradigm for Optical Imaging: Temporally Encoded Maps of Intrinsic Signal.</a> Neuron. 38, 529-545 (2003).</li><li>Llano, D. A., Turner, J., Caspary, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+cortical+inhibition+in+an+aging+mouse+model+of+chronic+tinnitus.">Diminished cortical inhibition in an aging mouse model of chronic tinnitus.</a> J. Neurosci. 32, 16141-16148 (2012).</li><li>Ehret, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+studies+on+auditory+development+in+mammals+in+relation+to+higher+nervous+system+functioning.">Behavioural studies on auditory development in mammals in relation to higher nervous system functioning.</a> Acta otolaryngol. 99, 31-40 (1985).</li><li>Mikaelian, D., Ruben, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+hearing+in+the+normal+CBA-J+mouse:+correlation+of+physiological+observations+with+behavioral+responses+and+with+cochlear+anatomy.">Development of hearing in the normal CBA-J mouse: correlation of physiological observations with behavioral responses and with cochlear anatomy.</a> Acta. 59, 451-461 (1965).</li></ol>

The authors have nothing to disclose.

This protocol describes improvements upon a previously described colliculo-thalamocortical brain slice in p12-20 mouse to study information flow in the auditory system1. This method has a number of advantages over other, similar, brain slice preparations by retaining connections between more brain areas in a single slice, which gives investigators new tools to understand the interaction and interplay between auditory nuclei in the forebrain. There have been a few key modifications in this protocol, compared to...

감각 주연부와 열등한 둔덕 간의 정보의 실질적인 처리가 있지만 그것이 청각 피질에 도달하기 전에 청각 시스템에서, 상당한 추가 처리가있다. 우리는 처리가 수행하고 변환 뇌가 들어오는 감각 정보를 해석 할 수 있습니다 방법에 대한 때문에 작은 방법에 대해 거의 알고있다. 후각을 제외한 각각의 감각은 초기 신호가 피질 상승으로 감소 높은 충실도로 중계되는 신호에 주연 매우 유사한 조직을 갖는다. 피질은 더 들어오는 정보를 조절하는 하부 구조에 대한 전망을 보냅니다. 이 복잡한 시스템은 생체 내뿐만 아니라 시험관 제제의 수가 다양한 방식으로 연구되어왔다. 전, 모든 연결은 감각 입력을 제어하고 측정하면서, 연결의 집합을 조사하기 위해 연구를 가능하게 그대로주어진 영역에서 출력. 이 방법을, 강렬 복잡한 출력 상승을주고, 다른 감각 입력, 각성,주의를 포함하여 다른 입력의 큰 다양성없이 제어에 거의 없다. 시험관, 뇌 조각이 하나의 세트를 캡처 잘라왔다 연구자들은 자극과 구 심성 또는 뇌 영역의 다양한 평가를 할 수 있도록 예측, 또는 두 개의 연결된 뇌 영역의. 이들은 종종 시상에 입력 또는 시상과 대뇌 피질에 출력 하나가 2-5 보존 thalamocortical 또는 tectothalamic 중 하나 조각입니다. 이러한 제제는, 약물, 전기 및 optogenetic 조작 다양한 허용한다. 그러나 두 개의 뇌 영역으로, 이들은 주로 정보의 전송을 평가하고 시상 통과함에 따라 정보의 변화를 평가하는 기능이 부족하다. 또한 주목 변조 역할을 세망 시상 투영은 6-9 인 PR이 조각에 ESENT. 여기에서 우리는 어떻게 시상 게이트 및 필터 정보의 독특한 관점을 제공하기 위해 시상에 다양한 입력의 연구자를 제어 할 수 있습니다 이전 준비 1시에 개선을 보여줍니다. 우리 커플 단일 세포 수준에서 다양한 입력의 영향을 측정하기 위해 슬라이스 연결 및 대규모 활성화 분석 뉴런 집단 분석 시상 칼슘 이미징 및 단일 세포 기록을 평가하기위한 flavoprotein 자기 형광 영상이 신규 슬라이스 제제. 이러한 광범위한 연결을 유지하는 데 도움을주기 위해 우리는 또한 강화 된 관류에 대한 슬라이스를 상승시키는 대신에 뇌 조각과 다리를 유지하는 일반 슬라이스 앵커 (일명 &quot;하프&quot;)의 수정을 개발했다. 하프는 슬라이스를 둘러싸고 하프 문자열에 대한 사용자 지정 부착 점 수 있도록 수정 된 말굽 모양으로 설계되어 있습니다. 세 개의 문자열은첨부 ⅰ) 하나 ⅱ) 하나가 AC의 꼬리 가장자리 IC의 꼬리 에지로부터 연장 ⅲ) 하나의 영역에 슬라이스의 내측 가장자리로부터 비스듬하게 연장되어, 슬라이스의 내측 가장자리를 따라 가로로 놓여 지도록 AC에 주동이 (도 1a 참조). 문자열 슬라이스 무결성을 유지할 수 있도록 슬라이스 압력 저하 량을 허용 하프 (시아 노 아크릴 레이트 접착제)을 붙이는 프레임에 작은 요철이 (도 1b 참조). 입체 인쇄를 사용하여, 우리는 조직의 상하 인공 뇌척수액 (ACSF)의 이상적인 흐름 수 있도록 우리 고유의 사양뿐만 아니라 다리에 사용자 정의 디자인 수금 할 수 있습니다. 이것은 또한 패치 클램프 전기 생리학의 조직에 침투하는 빛이 넓은 영역을 유지합니다.

<table><tbody><tr><td>High sucrose cutting solution</td><td></td><td></td><td>in mM: 206 sucrose, 10.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 11.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 0.5 CaCl&lt;sub&gt;2&lt;/sub&gt;, 2.5 KCl, pH 7.4</td></tr><tr><td>Low calcium aCSF</td><td></td><td></td><td>in mM: 126 NaCl, 3.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 10.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 1.0 CaCl&lt;sub&gt;2&lt;/sub&gt;, 2.5 KCl, pH 7.4</td></tr><tr><td>aCSF</td><td></td><td></td><td>in mM: 126 NaCl, 2.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 10.0 glucose, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 26 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 2.0 CaCl&lt;sub&gt;2&lt;/sub&gt;, 2.5 KCl, pH 7.4</td></tr><tr><td>Stimulus Isolator</td><td>World Precision Instruments</td><td>A360</td><td></td></tr><tr><td>DMSO</td><td>Life Technologies</td><td>D12345</td><td>Lot: 1572C502</td></tr><tr><td>Fura-2AM</td><td>Life Technologies</td><td>F1201</td><td>Lot: 144912</td></tr><tr><td>Pluronic F-127</td><td>Life Technologies</td><td>P3000MP</td><td>Lot: 1499369</td></tr><tr><td>Large culture dish</td><td>Fisherbrand</td><td>08-757-13</td><td>100 x 15 mm culture dish</td></tr><tr><td>Small culture dish</td><td>Falcon</td><td>353001</td><td>35 x10 mm culture dish</td></tr><tr><td>Raised culture membrane</td><td>Millicell</td><td>PICMORG50</td><td>Used to maintain oxygenated fluid perfusion on both sides of slice.</td></tr><tr><td>Flavoprotein imaging fluorescence cube</td><td>Olympus</td><td>UMNIB</td><td>470&amp;ndash;490 nm excitation, 505 nm dichroic, 515 nm emission long pass.&amp;nbsp; We have found that virtually any green fluorescence protein filter cube will work here.</td></tr><tr><td>Calcium imaging fluorescence cube</td><td>Omega Optical</td><td>BX-18</td><td>XF1005 365 nm exitation, XF2001 400 nm dichroic, XF3080 510 nm emission</td></tr><tr><td>Agar for blocking brain</td><td></td><td></td><td>3% by weight in water</td></tr><tr><td>Viper si Stereo Lithography Apparatus</td><td>3D Systems</td><td></td><td></td></tr></tbody></table>

modification of a colliculo-thalamocortical mouse brain slice, incorporating 3-d printing of chamber components and multi-scale optical imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

P15 마우스에서 얻은 colliculo-thalamocortical 마우스 뇌 조각의 예는 그림 2에 나타나있다. 이상적인 슬라이스 IC가 자극 될 때 모든 활성화 된 네 개의 주요 중뇌와 전뇌 청각 구조 IC, MGB, TRN, 및 AC를 포함합니다 (도 2A). 푸리에 분석을 사용하여, 상기 스펙트럼 전력은 주기적으로 자극 주파수 (10)에 혼입 인 활성을 나타내는 접속 뇌 영역과, 전기 자극의 주파수에서 측정...

Watch this Scientific Journal Video about Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging at JoVE.com

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

여러 각도를 사용하여 마우스 강아지의 머리를 잘라, 우리는 주요 청각 중뇌와 전뇌 구조의 가장 사이의 연결을 캡처 이전에 설명 급성 뇌 조각을 개선.

Colliculo-thalamocortical 마우스 뇌 조각의 변경, 3 차원 챔버 구성 요소의 인쇄 및 멀티 스케일 광학 이미징을 통합

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

modification-colliculo-thalamocortical-mouse-brain-slice

Research

JoVE Journal

Neuroscience

8.2K 조회수.  University of Illinois Urbana-Champaign.  여러 각도를 사용하여 마우스 강아지의 머리를 잘라, 우리는 주요 청각 중뇌와 전뇌 구조의 가장 사이의 연결을 캡처 이전에 설명 급성 뇌 조각을 개선. 

비디오: Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Thinned - 해골 준비를 사용하여 마우스 시각 피질의 만성 이미징

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

연구

신경과학

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

세미 두께 뇌 조각의 고해상도 형광 이미징에 대한 빠른 접근

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

3D 이미징 및 형태학의 분석을위한 성인 마우스 뇌혈관 캐스팅

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

다층 피질 칼슘<sup&gt; 2+</sup&gt; 프리즘 프로브 및 소형 형광 현미경으로 자유롭게 움직이는 마우스에서 이미징

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

행동분석학

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

마우스 뇌의 장기 이미징을위한 세련된와 철근 Thinned-해골 창

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

공 초점 빛 시트 현미경 전체 마우스 두뇌의 마이크론 규모의 해상도 광학 단층 촬영

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

정확 하 게 지역화 하 고 반복적인 뇌 조각 동봉 된 시스템에서의 장기적인 문화 중 간헐적인 이미징 수정된 롤러 튜브 메서드

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

마우스 피 질 세포 조직의 대형 3-차원 영상

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

3D Scanning Technology Bridging Microcircuits and Macroscale Brain Images in 3D Novel Embedding Overlapping Protocol

The human brain, being a multiscale system, has both macroscopic electrical signals, globally flowing along thick white-matter fiber bundles, and microscopic neuronal spikes, propagating along axons and dendrites. Both scales complement different aspects of human cognitive and behavioral functions. At the macroscopic level, MRI has been the current standard imaging technology, in which the smallest spatial resolution, voxel size, is 0.1&#8211;1 mm3. Also, at the microscopic level, previous physiological studies were aware of nonuniform neuronal architectures within such voxels. This study develops a powerful way to accurately embed microscopic data into a macroscopic map by interfacing biological scientific research with technological advancements in 3D scanning technology. Since 3D scanning technology has mostly been used for engineering and industrial design until now, it is repurposed for the first time to embed microconnectomes into the whole brain while preserving natural spiking in living brain cells. In order to achieve this purpose, first, we constructed a scanning protocol to obtain accurate 3D images from living bio-organisms inherently challenging to image due to moist and reflective surfaces. Second, we trained to keep speed to prevent the degradation of living brain tissue, which is a key factor in retaining better conditions and recording more natural neuronal spikes from active neurons in the brain tissue. Two cortical surface images, independently extracted from two different imaging modules, namely MRI and 3D scanner surface images, surprisingly show a distance error of only 50 &#956;m as mode value of the histogram. This accuracy is comparable in scale to the microscopic resolution of intercellular distances; also, it is stable among different individual mice. This new protocol, the 3D novel embedding overlapping (3D-NEO) protocol, bridges macroscopic and microscopic levels derived by this integrative protocol and accelerates new scientific findings to study comprehensive connectivity architectures (i.e., microconnectome).

This article introduces an experimental protocol using 3D scanning technology bridging two spatial scales: the macroscopic spatial scale of whole-brain anatomy imaged by MRI at &#62;100 &#956;m and the microscopic spatial scale of neuronal distributions using immunohistochemistry staining and a multielectrode array system and other methods (~10 &#956;m).

3D 소설 포함 프로토콜에서 마이크로 회로 및 매크로 스케일 뇌 이미지를 연결하는 3D 스캐닝 기술

The human brain, being a multiscale system, has both macroscopic electrical signals, globally flowing along thick white-matter fiber bundles, and ...

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

중시경에서 현미경 비늘에 이르기까지 신경 세포 이미징을위한 조직 제거 방법

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...