우리는 그래픽 제임스 Perna 감사합니다. 이 작품은 YZ에 정신 건강의 국립 연구소에서 교부금에 의해 지원되었다

승인은 수술 및 이미징 연구의 개시 전에 홈 기관에서 얻을 수 있어야합니다. 이 논문에 기술 된 실험은 캘리포니아 대학 산타 크루즈 기관 동물 관리 및 사용위원회의 지침과 규정에 따라 수행되었다. 1. 수술 <ol><li> 모든 수술 도구를 압력솥 철저하게 수술 전 70 % 알코올로 작업 영역을 소독. </li><li> KX 마취 용액 (200 ㎎ / kg 케타민 20 ㎎ / kg 자일 라진)을 복강 내 (IP) 주사하여 마우스를 마취 마우스의 체중에 따라 주 :. KX 투여는 건강 상태는 스트레인, 연령에 따라 조절 될 수 있고, 마우스. 최적의 복용량을 결정하는 수의 상담도 추천합니다. </li><li> 마우스의 발가락을 눌러 마취 상태를 모니터링 할 수 재귀 응답을 확인하여 주기적으로 발가락 핀치 테스트를 수행합니다.마우스가 완전히 수술을 시작하기 전에 마취되어 있는지 확인합니다주의 :. 장시간 영상 세션 동안, 필요한 경우 추가 KX를 리다, 주기적으로 마우스의 마취 상태를 확인합니다. </li><li> 수술 동안 체온을 유지하기 위해 가열 패드에 마우스를 배치. </li><li> 두피를 노출하는 면도날을 사용하여 쥐의 머리를 면도. </li><li> 알코올 패드와 베타 딘을 교대로 피부를 닦아 면도 영역을 소독. 잔여 머리를 잘라낸를 제거합니다. </li><li> 조심스럽게 따라서 실험 기간 동안 탈수로 인한 영구적 인 손상을 방지, 두 눈을 윤활 눈 연고를 적용합니다. </li><li> 두피의 중간 선을 따라 직선 절개를 확인하고, 두개골의 가장자리를 향해 옆으로 피부를 이동합니다. </li><li> 두개골에 부착 된 결합 조직을 제거합니다. </li></ol> 2. 시닝 두개골 준비 <ol><li> STER에 근거 촬상 영역을 식별. eotaxic 좌표 참고 : 큰 혈관, 블록 등의 침투를 방지하고 몇 군데 구조를 흐리게하십시오. 두개골은 멸균 식염수 촉촉한 때 혈관이 최고의 관찰된다. </li><li> 두개골 (0.5 ~ 1.0 mm 직경)의 얇은 원형 영역에 고속 마이크로 드릴을 사용합니다. 오히려 두개골에 대해 그것을 잡고 아래로 눌러보다 두개골 표면에 드릴 평행 이동합니다. 드릴은 외부 컴팩트 뼈의 층과 중간 해면골 층까지 모두 제거됩니다 참고 :. 과열로 인한 손상을 방지하려면, 드릴 비트와 두개골 사이의 장기간의 접촉을 피할 것. </li><li> 드릴 얇게 지역의 중심에 미세 수술 블레이드 내부 컴팩트 뼈 층을 얇게 계속합니다. 균등 대략의 두께로 작은 영역 (200 ~ 300 μm의 직경)를 얇게 할 때까지 머리에 아래로 누르지 않고 두개골을 긁어 약 45 °의 각도로 미세 수술 날을 잡고LY 20 ~ 30 μm의를 얻을 수있다. . 인해 두개골의 자동 형광으로, 두께는 이광자 현미경 주 미만의 두개골 상부 및 하부면 사이의 거리를 스캐닝함으로써 측정 될 수있다 : 20μm 미만의 두개골 두께가 좋은 화상 품질을 제공하지만 그것은 이후의 재 이미징 세션의 숱이 과정을 어렵게하기 때문에하지 않는 것이 좋습니다. 지나치게 숱이 방지하기 위해, 두개골의 두께는 (토론 참조) 수술하는 동안 정기적으로 점검 할 필요가있다. </li></ol> 3. 고정화 <ol><li> 조심스럽게 뼈 파편을 제거합니다. </li><li> 압력 가마로 소독 된 멸균 헤드 판의 중심 개구의 각 가장자리에 시아 노 아크릴 레이트 접착제의 작은 방울 (그림 1 참조)를 놓습니다. . 개구부의 중심 주에있는 얇아진 영역과 두개골에 단단히 머리 판을 잡고 : 접착제가 얇아진 연구를 오염 시키면사고로 egion, 미세 수술 칼날로 조심스럽게 제거합니다. </li><li> 부드럽게 헤드 판의 중심 개구의 가장자리에 두개골의 측면으로부터 피부를 당겨. </li><li> 헤드 플레이트가 아니라 두개골에 부착 될 때까지 약 10 분을 기다립니다. </li><li> (그림 1 참조) 보유 판의 두 가지 측면 블록 헤드 플레이트를 위치하게 한 후, 고정 플레이트에 마우스를 고정시키기 머리 판의 가장자리에 나사를 조입니다 참고 :. 사이에 피부 나 수염이 없는지 확인합니다 나사를 조이기 전에 머리 판과 블록. 동물의 뒤 부드럽게 쓰다듬어 때 양호한 고정화 제제 해부 현미경 두개골 전혀 관찰 움직임을 표시 안된다. </li><li> . 미 중합 접착제를 제거하기 위해 식염수로 노출 된 두개골을 씻어 주 : 미 중합 접착제 이미지를 흐리게하고 손해 목표를 현미경으로 그것은 남아있는 접착제를 제거하는 것이 중요합니다 &lt;./ 리&gt; </ol> 4. 이미징 <ol><li> (그림 2 참조) 이후의 영상 세션에서 몇 군데 지역을 재배치하는 데 사용되는지도 1로 희석 영역과 노출 된 두개골의 혈관의 사진을 가져 가라. </li><li> 영상 현미경으로 마우스를 놓습니다. 표면 형광을 사용하여 10X 공기 목적에 따라 얇아진 영역을 찾아보기의 중앙에 얇은 영역을 이동합니다. </li><li> 두개골의 상단에 식염수 한 방울을 추가하고 멸균 수로 세척 된 60X 목표로 전환. 각각의 돌기 쪽이 명확하게 수상 돌기를 따라 시각화하는 지역을 선택합니다. 식별 및 epifluorescent보기와 혈관의 사진 사이에 혈관을 비교하여지도 1에 해당하는 지역을 라벨. </li><li> 형광에 따른 튜닝 이광자 레이저 파장. 예를 들어, YFP 920 nm의; GFP에 대한 890 nm의; DsRed를하고 tdTomato 24 1,000 nm의. </li><li> 2 이미지 스택 획득60X 목표를 사용하여 Z 축을 따라 μm의 단계. 이 이미지 스택은 약 200 ㎛ × 200 ㎛의 영역 (512 X 512 픽셀)를 커버하고 (그림 2 참조) 이후의 영상 세션에서 이전에지도 2로 사용됩니다. </li><li> 배 디지털 줌을 사용하여지도 2 내에서 아홉 이미지 스택을 획득. . 각각의 이미지 스택은 z 축 주에 따라 0.7 μm의 단계를, 70 μm의 X 70 μm의 (512 X 512 픽셀)의 대략적인 지역을 커버 : 광독성을 최소화하기 위해 샘플을 측정했을 때 레이저의 강도는 40 mW의 이하이어야한다. </li></ol> 5. 복구 <ol><li> 영상에 따라 부드럽게 두개골에서 머리 판을 분리합니다. </li><li> 철저하게 두개골과 나머지 모든 접착제를 제거하기 위해 피부를 청소 참고 :. 피부에 남아있는 접착제는 자극의 원인과 두개골에 남아있는 접착제는 두개골의 침식의 원인이됩니다 동안 피부의 치유 속도를 느리게합니다ngiogenesis 새로 성장 뼈의 층에서, 이후의 재배치 및 재 이미징 어려운 만들기. </li><li> 두개골과 생리 식염수를 여러 번로 피부를 씻어. </li><li> 무균 수술 봉합과 두피를 봉합. </li><li> 별도의 케이지에 가열 패드에있는 동물을 유지. 수술 후 통증을 완화하기 위해 피하 부 프레 노르 핀의 진통 (0.1 ㎎ / ㎏)을 관리 할 수​​ 있습니다. 전체 복구 홈 케이지에 동물을 반환합니다. 절개 부위가 치유되고 봉합이 제거 될 때까지 (적어도 하루에 한 번 확인) 밀접하게 동물을 모니터링합니다. 필요한 경우 부 프레 노르 핀 진통제의 후속 주사를 관리 할 수​​ 있습니다. </li></ol> 6. 재 이미징 <ol><li> 반복 1.1-1.8 단계를. </li><li> 반복 3.1-3.6 단계를 </li><li> 1지도하는 혈관 패턴과 2를지도하는 수지상 분기 패턴을 비교하여 이전에 몇 군데 지역을 찾습니다. </li><li> 재 이미징이 일주 내에서 수행되는 경우, 박형화 재 위에 새로 자란 뼈의 얇은 층을 제거하는 단계 2.3를 반복미세 수술 블레이드를 사용하여 기온. 재 이미징 이상 일주 한 후에 수행된다면, 단계 2.2 및 2.3를 반복 모두 참고. 새로 자란 뼈 층은 축소 화상 품질 선도 일본어 치밀골 층에 비해 더 적은 응집 구조로 구성된다. 따라서, 동일한 품질을 확보하기 위해서는 이전 촬상 세션보다 약간 얇은 스컬하는 것이 필요하다. </li><li> 이전 이광자 현미경 영상 스택을 찍은 이미지와 일치 스택을 얻기 위해 위치 및 방향을 조정한다. </li><li> 단계 4.6에서와 같이 이미지를 획득. </li><li> 반복 영상 후 5.1-5.5 단계를. </li></ol>

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E., Zettel, M. L., Ison, J. R., Allen, P. D., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+aging+and+sensory+loss+on+glial+cells+in+mouse+visual+and+auditory+cortices.">Effects of aging and sensory loss on glial cells in mouse visual and auditory cortices.</a> Glia. 60, 541-558 (2012).</li><li>Lam, C. K., Yoo, T., Hiner, B., Liu, Z., Grutzendler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embolus+extravasation+is+an+alternative+mechanism+for+cerebral+microvascular+recanalization.">Embolus extravasation is an alternative mechanism for cerebral microvascular recanalization.</a> Nature. 465, 478-482 (2010).</li><li>Kelly, E. A., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+imaging+of+mouse+visual+cortex+using+a+thinned-skull+preparation.">Chronic imaging of mouse visual cortex using a thinned-skull preparation.</a> J Vis Exp. , (2010).</li><li>Marker, D. F., Tremblay, M. E., Lu, S. M., Majewska, A. K., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin-skull+window+technique+for+chronic+two-photon+in+vivo+imaging+of+murine+microglia+in+models+of+neuroinflammation.">A thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation.</a> J Vis Exp. , (2010).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Feng, G., 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> J Vis Exp. , (2012).</li><li>Zhang, L., 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=Imaging+glioma+initiation+in+vivo+through+a+polished+and+reinforced+thin-skull+cranial+window.">Imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window.</a> J Vis Exp. , (2012).</li><li>Pacary, E., 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=Visualization+and+genetic+manipulation+of+dendrites+and+spines+in+the+mouse+cerebral+cortex+and+hippocampus+using+in+utero+electroporation.">Visualization and genetic manipulation of dendrites and spines in the mouse cerebral cortex and hippocampus using in utero electroporation.</a> J Vis Exp. , (2012).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental biology. 240, 237-246 (2001).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+injection+of+adeno-associated+viral+vectors.">Intracranial injection of adeno-associated viral vectors.</a> J Vis Exp. , (2010).</li><li>Taniguchi, H., 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=A+resource+of+Cre+driver+lines+for+genetic+targeting+of+GABAergic+neurons+in+cerebral+cortex.">A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.</a> Neuron. 71, 995-1013 (2011).</li><li>Zariwala, H. A., 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=A+Cre-dependent+GCaMP3+reporter+mouse+for+neuronal+imaging+in+vivo.">A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.</a> J Neurosci. 32, 3131-3141 (2012).</li><li>Kuhlman, S. J., Huang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+labeling+and+functional+manipulation+of+specific+neuron+types+in+mouse+brain+by+Cre-activated+viral+gene+expression.">High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.</a> PloS one. 3, (2008).</li></ol>

저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

성공적인 얇게 두개골 준비를 얻으려면,이 프로토콜의 몇 가지 단계가 매우 중요하다. 1) 두개골의 두께. 두개골 뼈이 고밀도 치밀골의 층과 저밀도 해면골의 중간층과 함께 샌드위치 구조를 갖는다. 고속 마이크로 드릴 치밀골과 해면골의 외부 층을 제거하기에 적합한 반면, 미세 수술 블레이드 치밀골의 내부 층을 얇게 이상적이다. 개발하는 동안 두개골의 두께가 증가하고 강성으로, 성인 쥐의...

포유류의 피질 감각 지각과 운동 제어에서 추상적 인 정보 처리 및 인식, 많은 뇌 기능에 참여하고 있습니다. 다양한 대뇌 피질의 기능은 신경 세포의 다른 종류의 통신 및 개별 시냅스에서 정보를 교환하기로 구성되어 다른 신경 회로에 구축 할 수 있습니다. 시냅스의 구조 및 기능은 일관 경험 및 병리에 대한 응답으로 수정되고있다. 성숙한 뇌에서 시냅스 가소성 형성과 기능적 신경 회로의 유지에 중요한 역할을 수행, 강도 변화와 시냅스의 추가 / 제거를 두의 형태를 취한다. 돌기 쪽은 포유류 뇌의 흥분성 시냅스의 대부분의 시냅스 구성 요소입니다. 지속적인 매출 및 척추의 형태 학적 변화는 시냅스 연결 1-7 수정의 좋은 지표 역할을 것으로 추정된다. 이광자 레이저 스캐닝 마이크로SCOPY 두꺼운 불투명 준비하고 그대로 뇌 8 라이브 영상에 적합하게 낮은 광독성을 통해 깊은 침투를 제공합니다. 형광 표시와 함께, 두 광자 영상은 살아있는 뇌 들여다 높은 공간과 시간적 해상도를 가진 개별 시냅스의 구조 개편을 수행 할 수있는 강력한 도구를 제공합니다. 다양한 방법은 실시간 이미징 9-13 생쥐를 제조하는데 사용되었다. 여기, 우리는 마우스 피질에서 시냅스 돌기 쪽의 구조적 가소성을 조사하기 위해 생체 내 두 광자 이미징 얇게 두개골 준비에 대해 설명합니다. 이 방법을 사용하여, 우리의 최근의 연구는 형광 표지 된 신경 세포의 하위 집합 및 생체 표지 기술의 급속한 발전과 함께 형질 전환 동물의 증가 가용성 학습 운동 기능에 대한 응답으로 돌기 척추 변화의 동적 인 그림을 묘사 한, 여기에 설명 된 절차와 유사한 절차도 적용 할 수 있습니다 investiga하기TE 다른 세포 유형 및 피질 영역, 다른 조작과 결합뿐만 아니라, 16-23은 질병 모델에서 사용했다.

<table border="0" cellpadding="0" cellspacing="0" width="1253">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ketamine</td>
			<td style="width:181px;">Bioniche Pharma</td>
			<td style="width:108px;">67457-034-10</td>
			<td style="width:775px;">Mixed with xylazine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Xylazine</td>
			<td style="width:181px;">Lloyd laboratories</td>
			<td style="width:108px;">139-236</td>
			<td>Mixed with ketamine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saline</td>
			<td style="width:181px;">Hospira</td>
			<td style="width:108px;">0409-7983-09</td>
			<td>0.9% NaCl for injection and imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Razor blades</td>
			<td style="width:181px;">Electron microscopy sciences</td>
			<td style="width:108px;">72000</td>
			<td>Double-edge stainless steel razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Alcohol pads</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">06-669-62</td>
			<td>Sterile alcohol prep pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Eye ointment</td>
			<td style="width:181px;">Henry Schein</td>
			<td style="width:108px;">102-9470</td>
			<td>Petrolatum ophthalmic ointment sterile ocular lubricant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">High-speed micro drill</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">18000-17</td>
			<td>The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Micro drill steel burrs</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">19007-14</td>
			<td>1.4 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Microsurgical blade</td>
			<td style="width:181px;">Surgistar</td>
			<td style="width:108px;">6961</td>
			<td>The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Cyanoacrylate glue</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">NC9062131</td>
			<td>Fix the head plate onto the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Suture</td>
			<td style="width:181px;">Havard Apparatus</td>
			<td style="width:108px;">510461</td>
			<td>Non-absorbale, sterile silk suture, 6-0 monofilament</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Dissecting microscope</td>
			<td>Olympus</td>
			<td style="width:108px;"></td>
			<td>SZ61</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Two-photon microscope</td>
			<td style="width:181px;">Prairie Technologies</td>
			<td style="width:108px;"></td>
			<td style="width:775px;">Ultima IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">10X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 0.30, air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">60X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 1.1, IR permeable, water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ti-sapphire laser</td>
			<td style="width:181px;">Spectra-Physics</td>
			<td style="width:108px;"></td>
			<td>Mai Tai HP</td>
		</tr>
	</tbody>
</table>

two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

포유류의 피질에서 신경 세포는 매우 복잡한 네트워크와 시냅스에서 정보 교환을 형성한다. 시냅스 강도뿐만 아니라, 시냅스의 추가 / 제거의 변화, 신경 가소성의 구조적 기초를 제공하고, 환경 의존적으로 발생한다. 피질에서 가장 흥분성 시냅스의 시냅스 구성 요소로, 돌기 쪽은 시냅스의 좋은 프록시로 간주됩니다. 마우스 유전학과 형광 라벨 기법, 개별 뉴런과 자신의 시냅스 구조의 촬영 장점은 그대로 뇌에 표시 할 수 있습니다. 여기에서 우리는 생체 내에서 시간이 지남에 형광 표지 시냅스 돌기 쪽을 따라 두 광자 레이저 스캐닝 현미경을 사용하여 두개 영상 프로토콜을 소개합니다. 이 프로토콜은 그대로 두개골을 유지하고 수막의 노출과 피질에 의한 염증 효과를 방지 얇게 두개골 준비를 활용합니다. 따라서, 이미지는 SU 직후에 취득 할 수있다rgery이 수행된다. 실험 절차는 시간부터 년까지 다양한 시간 간격들에 걸쳐 반복하여 수행 될 수있다. 이 혼합물을 또한 생리적 및 병리 적 조건에서, 다른 피질 영역과 층뿐만 아니라, 다른 세포 유형을 조사하기 위해 확장 될 수있다.

YFP-H 라인 쥐 25에서 노란색 형광 단백질이 대뇌 피질의 표면 층에 자신의 혀끝의 수상 돌기를 돌출 레이어 V 피라미드 신경의 부분 집합에 표현한다. 얇게 두개골 준비를 통해, 형광 표지 된 수지상 세그먼트 반복적 시간에서 달에 이르기까지, 다양한 영상 간격을 통해 두 광자 현미경으로 몇 군데 있습니다. 여기에서 우리는 개인의 척추뿐만 아니라 filopodia 명확하게 수상 돌기를 따라 시?...

Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

두 광자<em&gt; 생체</em&gt; 시닝 두개골 준비를 사용하여 마우스 피질 돌기 쪽의 영상

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

two-photon-vivo-imaging-dendritic-spines-mouse-cortex-using-thinned

Research

JoVE Journal

Neuroscience

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

17.8K 조회수.  University of California, Santa Cruz. Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

비디오: Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

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 ...

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신경과학

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

두 광자 현미경을 사용하여 마우스 척수의 생체내 이미징에

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

내생 NADH 형광의 2 광자 이미징에 의한 마우스 대뇌 피질의 Microregional Hypoxia의 탐지

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작<em&gt; utero에서</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

에 얇게 - 두개골 두피 창 기술<em&gt; 생체 내</em&gt; 광 결맞음 단층 촬영 영상

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

두피 뉴런에서 생체 내 두 - 광자의 영상은 경험에 의존 분자 변경

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

마우스 척수의 세포 역학의 두 광자 이미징

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

동시에 두 광자<em&gt; 생체</em&gt; 마우스 Retrosplenial 코어 텍스에서 시냅틱 입력의 이미징 및 시냅스 대상

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Vivo에서 신생아 쥐에 있는 대뇌 피 질의 뉴런의 두 광자 영상

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...