这项工作是由加拿大自然科学和工程研究理事会（NSERC）发现格兰特＃40352支持，校园艾伯塔省创新程序委员会主席，阿尔伯塔省老年痴呆症研究计划，以MHM，并NSERC CREATE在BIF博士生奖学金和AIHS研究生奖学金到MK。我们感谢浦明·沃对这个协议的发展和外科培训，并贝·米尔扎·阿加和狄鞘的养殖。

<html>下面的协议如下莱斯布里奇动物护理委员会（ACC）的指导方针的大学，并根据加拿大动物保护协会（CCAC）的标准进行的。 1.准备<ol><li>对于长期研究阶段，高压灭菌所有打开的手术用品，并确保不育是保持整个手术。如果需要多次手术，手术之间的反应釜。 </li><li>确保有充足的缓冲脑的手头上（至少50 mL）中。该溶液是由134 mM氯化钠，5.4 mM的钾，氯化镁1mM的六水合物，1.8mM的氯化钙二水合物，和5mM HEPES钠，pH 7.4平衡的用氯化-5 M氢。 </li><li>鼠标放置在感应室，并与3麻醉 - 4％异氟烷。 1.0请 - 2.0％异氟烷在手术过程中的维护。进一步降低到低至0.4 - 0.8％成像期间S = "外部参照"&gt; 5，13，14，15，提供了适当的麻醉维持。这些低麻醉水平要求的鼠标（约每5分钟）的警惕和频繁的监测，以确保它仍然areflexic对疼痛刺激。 注:脱水可由于高的表面成为问题鼠标的体积比和长时间使用异氟醚的加剧。 <ol><li>使用盐水的皮下注射，每10g体重0.1mL的，每1 - 2小时。当充分地水合，鼠标会小便一次，每1 - 2小时。 </li></ol></li><li>密切监视鼠标，以确保整个手术和成像一致麻醉。不要离开鼠标无人值守和照顾它永远不会恢复意识。 </li><li>传送麻醉小鼠在头保持器设置和地点上设置为37℃的热调节的加热垫。安全上最佳匹配的在齿架小时。 </li><li>旋转鼠标的向左头约30°，以暴露所述头部的右横向侧并与耳棒（ 图1A）的钝端固定小鼠的头部。 </li><li>应用眼药膏，以防止角膜干燥。 </li><li>为了减少脑水肿，（4毫克/千克）肌内注射地塞米松。 </li><li>抹在4％氯己定（3次）和70％乙醇（3次）浸渍棉签手术区域的皮肤上。每次使用棉签只有一次。 </li><li>唐外科手套并覆盖有粘合剂保鲜膜的动物。由利多卡因注射提供局部麻醉剂（8 - 10毫克/千克; 2％肾上腺素）皮下在开颅手术部位。等待3 - 5分钟，使药物被吸收到组织中。 </li></ol> 2.取出皮肤和头骨缩回肌肉<ol><li>而下一个观察颅骨执行几乎所有的这些程序解剖显微镜（ 例如 0.7-4.5倍的功率，视情况而定）。 </li><li>用镊子提起中线左侧1毫米（仅在耳后），并用手术剪刀做一个小的水平切口。 </li><li>对右耳进行5 - 6 mm的横向切割，然后朝头部的头端切开。 </li><li>在初始切口处，插入剪刀，并沿切线10毫米。 </li><li>切割右耳和右眼附近的皮肤，以暴露头骨和颞肌的右侧。确保暴露区域的最宽部分至少为7 mm。如果手术区域需要延长，请进一步修剪皮肤。 </li><li>通过在颅骨和皮肤之间放入几滴氰基丙烯酸丁酯胶来固定切口周围的皮肤。确保组织已经预先用棉花涂抹器或滚动的组织干燥以增加粘合剂的功效。 </li><li>用棉签擦拭表面以圆周运动的头骨移除从颅骨骨膜。确保不仍然是完全干燥的头骨。 </li><li>使用弹簧剪刀和镊子，分离从头骨的颞肌;切割和横向缩回肌肉直至到达鳞骨（ 图1C）。要格外小心，不要损坏沿靠近眼睛的鳞骨的水平运行颞浅静脉，否则赔了可能发生。 </li><li>控制用凝胶泡沫预浸泡在脑出血缓冲器。对于严重的出血使用热cauterizer。降丁基氰基丙烯酸酯胶到出血部位以下与热cauterizer治疗。确保的区域是通过使用棉尖涂药器或轧制组织以增加粘合剂的效力预先干燥。 </li></ol> 3.开颅手术注:切除颅骨的过程中，外科医生必须保持勤奋和硬脑膜，以避免不必要的复杂性。包括故障排除步骤应该出现并发症。 <ol><li>标记前囟的任用细尖标记或切一小三角形片胶带并在前囟点的角部（ 图2A）来定向底层脑区的位置。 </li><li>之前固定所述头板确保颅骨完全干燥。颅骨将快速风干脑缓冲器浸泡凝胶泡沫的应用之后，如果需要更多的干燥，可使用棉尖拭子。这个步骤是用于头板固定（ 图1B）是至关重要的。 注意:如果头骨骨膜左，或者如果头骨是不是粘在头上盘前干的，它可能会脱落。如果发生这种情况，温和地去除头板，然后重新开始。出血在此过程中，可能会发生;允许几分钟它凝结，然后轻轻取出。不推荐使用此过程要重复两次以上。 </li> &lt;LI&gt;应用乙基氰基丙烯酸酯胶围绕头部板16的底部边缘，和胶水头板在开颅手术区域（ 图1D和图2A）。 </li><li>与牙科水泥只剩下开颅手术区域暴露填补了头骨和头板之间的开口空间。等待牙科用粘固剂干燥和硬化，通常为5 - 10分钟（ 图2B）。 <ol><li>一旦水泥设置，简单地用脑缓冲填充井，并允许浸泡3 - 5分钟。使用卷纸钻孔（ 图2C）之前去除脑缓冲器。 </li></ol></li><li>通过轻轻颅骨的表面与牙钻得分勾勒出手术区域。使用风钻（设置为最大20 PSI的），具有FG¼毛刺，并用一个可变速度脚踏板控制的。 </li><li>轻轻地追查沿原得分钻深化它，E确保钻头不会穿过颅骨进入大脑（ 图2D ）。每隔几分钟轮流，钻孔和用蘸有滚烫的组织颅骨表面。这将减少颅骨的加热和干燥从机械摩擦和长时间的暴露。 注意:使用湿凝胶泡沫后，颅骨将迅速空气干燥。如果需要更多干燥，请使用棉签拭子。 注意:头骨厚度不均匀。例如，顶叶嵴是最厚的区域，而中线附近的头骨区域和鳞状的地标相对较薄。 </li><li>在钻孔期间，通过用镊子或不动钻头轻轻地按压颅骨，定期检查颅骨的屈曲。当骨头开始弯曲时，停止钻孔并将整个窗口浸入脑缓冲液中。 注意:如果血液从一个区域冲出，可能表明硬脑膜已被损坏。如果是这样的话，放一个semI-湿区域上方凝胶泡沫，并尝试以吸收血液，同时轻轻用棉拭子末端将压力施加到凝胶泡沫。 </li><li>等待至少5分钟，头骨移除之前软化骨并减少硬脑膜粘到骨的机会，使得头骨移除过程更容易。 </li><li>而颅骨脑缓冲淹没执行头骨移除过程。 注意:如果该头骨的一部分保持附着顽固，一个＃11手术刀刀片可用于轻轻得分颅骨。要格外小心不要通过头骨到大脑穿刺刀。 </li><li>从前缘开始，轻轻撬动从使用镊子硬脑膜松散头骨。 注:如果在头骨移除过程中发生出血的量小，用移液管或注射器去除缓冲液，然后用新的缓冲区替换。 </li><li>一旦骨松动和硬脑膜"浮动"，紧紧握住用钳子骨和抬离骨硬脑膜。确保骨从来没有渗透到大脑。 </li><li>为了控制出血，滚组织的角落成一点，并从颅很好地去除大部分的缓冲区。快速应用凝胶泡沫，预先浸泡在缓冲液中，于出血部位，同时用棉签擦拭添加很轻的压力。 注:出血通常来自于骨或硬脑膜的表面的边缘;这两种情况都是正常的，如果没有大的血管受损出血会迅速停止。如果继续出血，血液可以填充整个窗口，形成覆盖所述成像区域中的凝块片。 <ol><li>以除去凝块片，小心地同时使血块周围的血管破裂的源完好从摄像区域拾取的凝结血块。注意不要从泄放源取出血块，因为这可能会导致更多的血液流失。灌溉大脑表面脑缓冲洗去任何血液。 </li><li>小心避免接触细腻的脑组织或添加异物到大脑;重复，直到出血停止，约2 - 5分钟（ 图2E）。 注:在这一点上，开颅手术准备准备颅窗口（见第5步）。如果需要，植入颅窗口（参见步骤4）之前，除去硬脑膜。 </li></ol></li></ol> 4.去除杜拉注:杜拉过程需要格外小心，可能需要超过15分钟。 <ol><li>从开颅客场战平多余的缓冲液。同时保持湿润表面，抓住一小块硬脑膜用镊子轻轻撕硬脑膜。 </li><li>使用镊子和剪刀春天轻轻撕开，并切去硬脑膜。 </li><li>降脑表面上更多的大脑缓冲浮动硬脑膜，并帮助它从大脑中分离出来。直到所有硬脑膜从颅窗网站上删除。当正确执行大脑就会显得非常干净的具有鲜明BLOOD血管和没有瑕疵（比较2F 图2E）。 注意:硬脑膜的某些区域连接到小动脉脑（ 例如 ，中线附近靠近颅顶联合区）的表面，去除这种可破裂动脉。在这种情况下，它可能是最好保留一小块硬脑膜完好在动脉之上的。该VSD可能无法穿透小面积，但是这是优选的有大出血。 </li><li>尽快从脉动最小化运动，并进一步防止肿胀固定在琼脂糖脑表面（参见步骤5）。 </li></ol> 5.准备颅窗<ol><li>喷雾盖玻璃用70％乙醇，并使用空气罐轻轻地干燥。确保玻璃与没有斑点或灰尘存在完全清洁。 </li><li>通过加热200毫克琼脂糖粉末溶解在15毫升脑缓冲器的制备1.3％的琼脂糖。设置microwAVE到高和热为10 - 15秒的时间，之间温和搅拌中，直到所有的琼脂溶解。 注意:确保没有气泡或颗粒存在，因为这些将干扰成像。 </li><li>放置在热琼脂糖温度计和冷却琼脂糖至刚好高于固化温度（〜40℃）。 注:在琼脂糖容器的外部运行冷水可加速冷却过程。轻轻搅拌连续，以确保没有气泡或颗粒存在。 </li><li>立即将琼脂之前，从颅很好地去除脑缓冲区。画了一个移液管的琼脂糖，并直接对大脑下降琼脂糖。快速放置在表面上的盖玻片固定琼脂糖盖玻片滴在了角落。 </li></ol> 6.安乐死注:根据我们的经验，这个过程需要经验的外科医生至少3 - 4实践手术达到大于90％成功率。缺乏经验的外科医生可能需要更多的实践。在开颅手术或硬脊膜切开，大脑可能蒙受损害，例如，如果通过骨到脑中钻拳。在开颅手术的边缘的一些轻微损坏可能是允许的。然而，如果大脑看起来并不"干净"与鲜红完好的血管和白色皮质，实验可能需要被终止。可怜的制剂的实例包括那些死者的血管，或在皮质上标有撕裂或破损的血管。如果这些迹象都存在，实验将不可能产生高质量的数据。无论手术/实验成功与否，人道在实验完成安乐死鼠标。 <ol><li>至少有3.5％的异氟烷​​麻醉深刻。然后，给予戊巴比妥钠的腹膜内注射300毫克/公斤。理想情况下，如果针插入到肝脏，死亡会非常快速（&lt;1分钟）。 </li><li>如果需要灌注，确认该动物是诉讼程序（见步骤1.3）之前，深度麻醉。 </li><li>可替代地，如果不需要灌注，等待至少5分钟，然后验证该小鼠是死的。确认没有呼吸和心跳，以及缺乏疼痛撤离及角膜反射消失。此外，观察四肢的淡蓝色/白色着色和变暗血管在皮层。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscale+transcranial+spontaneous+activity+mapping+in+GCaMP3+transgenic+mice+reveals+extensive+reciprocal+connections+between+areas+of+somatomotor+cortex.">Mesoscale transcranial spontaneous activity mapping in GCaMP3 transgenic mice reveals extensive reciprocal connections between areas of somatomotor cortex.</a> J Neurosci. 34 (48), 15931-15946 (2014).</li><li>Xie, Y., 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=Resolution+of+High-Frequency+Mesoscale+Intracortical+Maps+Using+the+Genetically+Encoded+Glutamate+Sensor+iGluSnFR.">Resolution of High-Frequency Mesoscale Intracortical Maps Using the Genetically Encoded Glutamate Sensor iGluSnFR.</a> J Neurosci. 36 (4), 1261-1272 (2016).</li><li>Chan, A. W., Mohajerani, M. H., LeDue, J. M., Wang, Y. T., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscale+infraslow+spontaneous+membrane+potential+fluctuations+recapitulate+high-frequency+activity+cortical+motifs.">Mesoscale infraslow spontaneous membrane potential fluctuations recapitulate high-frequency activity cortical motifs.</a> Nat Commun. 6, 7738 (2015).</li><li>Lim, D. 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=In+vivo+Large-Scale+Cortical+Mapping+Using+Channelrhodopsin-2+Stimulation+in+Transgenic+Mice+Reveals+Asymmetric+and+Reciprocal+Relationships+between+Cortical+Areas.">In vivo Large-Scale Cortical Mapping Using Channelrhodopsin-2 Stimulation in Transgenic Mice Reveals Asymmetric and Reciprocal Relationships between Cortical Areas.</a> Front Neural Circuits. 6, (2012).</li><li>Ferezou, 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=Spatiotemporal+dynamics+of+cortical+sensorimotor+integration+in+behaving+mice.">Spatiotemporal dynamics of cortical sensorimotor integration in behaving mice.</a> Neuron. 56 (5), 907-923 (2007).</li><li>Mohajerani, M. H., Aminoltejari, K., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mini-strokes+produce+changes+in+interhemispheric+sensory+signal+processing+that+are+indicative+of+disinhibition+within+minutes.">Targeted mini-strokes produce changes in interhemispheric sensory signal processing that are indicative of disinhibition within minutes.</a> Proc Natl Acad Sci U S A. 108 (22), E183-E191 (2011).</li><li>Madisen, 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=Transgenic+mice+for+intersectional+targeting+of+neural+sensors+and+effectors+with+high+specificity+and+performance.">Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.</a> Neuron. 85 (5), 942-958 (2015).</li></ol>

作者什么都没有透露。

这对于大颅窗口创新协议使得能够在大脑皮质的时间和顶区同时成像。与光学成像结合，它可以帮助自发的和刺激诱发的活动中来揭示皮层区域内的神经动力学。这种膨胀的开颅手术还公开了一个大的扩展皮质脉管系统的网络，包括大脑中动脉（MCA）的近端，从而使在血流和血管外侧缺血性模型的直接操纵的体内成像。这种技术将是伟大的使用表达电压和钙指标蛋白23小鼠最近开发线。这些小鼠提供绕过对皮质培养电压敏感的染料需要的实用优势。这些外在染料需要时间来充分地渗入脑组织（〜60 - 90分钟），并通过温和毒性的限制。大开颅手术也有以前用来研究发育大鼠脑组织与VSDI 11。新生鼠具有更大的头部，并与成年老鼠的大小相当。这给予研究人员一个独特的机会，研究发展问题在神经科学中，虽然没有使用转基因小鼠。 这种方法的主要限制是无法慢性实验。颅骨的曲率使得钻井过程更具挑战性和时间小于开颅手术耗时。对于这种大开颅，至关重要的是在头部与中央缝合和鳞地标定位到平行于透镜的聚焦平面内。而大脑的一些失真从大脑的曲率预期，这些是由聚焦到皮质的表层克服。此问题是由获得刺激和平均的多次重复进一步缓解。总之，我们的大开颅技术被广泛应用licable为神经生物学当前问题的研究。

开颅是使用神经科学家以显示脑的一部分的标准程序。由于电的曙光，开颅手术已经允许在神经科学领域前所未有的突破。与电极大脑皮层的密集映射导致基于这些地图的实验测试假说和理论。我们最近进入其中开颅正用于皮质血流1，2，3和神经血管结构4的体内成像，使曝光区5，6，7内皮层活动的实时可视化的新时代。虽然许多研究使用与开颅体内光学成像技术相结合，研究结构和皮层神经元，神经胶质的功能，和COR蒂卡尔脉管系统8， 图9，进一步调查由暴露皮质的小区域限定（但参阅10）。 该协议的目的是提供一种用于产生大的横向开颅，从中线暴露大脑皮层到鳞骨，并且延伸超出前囟和lambda提供的方法。这种大的开颅使得关联皮层的同时观看（脾后，扣，和顶叶），原发性和继发性马达，体感，视觉和听觉皮层。这种方法已经被预先加上电压敏感染料成像（VSDI）调查皮层区域如何多个自发的和刺激诱发的皮质活动5，11，12中彼此交互。此过程中最具挑战性的方面包括定位头动物的，固定所述头板，和避免出血，同时从顶骨分离颞肌。护理也必须在钻井和头骨移除过程作为头骨曲线以倾斜的角度拍摄。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>Heating Pad&#160;</td>
			<td>FHC</td>
			<td>40-90-2</td>
			<td></td>
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			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
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		<tr>
			<td>Forceps&#160;</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>2 or more pairs are recommended</td>
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			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00, 15000-10</td>
			<td>1 pair should be designated for dura removal&#160;</td>
		</tr>
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			<td>Jet tooth shade powder</td>
			<td>LANG Dental</td>
			<td>Jet Tooth Shade Powder</td>
			<td>to be mixed with the Jet Liquid</td>
		</tr>
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			<td>Jet tooth shade liquid</td>
			<td>LANG Dental</td>
			<td>Jet Tooth Shade Liquid</td>
			<td>to be mixed wihth the Jet Powder&#160;</td>
		</tr>
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			<td>Drill Heads - Carbide Burs FG 1/4 389</td>
			<td>Midwest Dental</td>
			<td>385201</td>
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			<td>Sigma-Aldrich</td>
			<td>A9793</td>
			<td></td>
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			<td>Gelfoam</td>
			<td>Sinclair Dental Canada</td>
			<td>Pfizer Gelfoam</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>124125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine 2% Epinephrine</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>125299</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethazone 5 mg/mL</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>125231</td>
			<td></td>
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			<td>Butyl cyanoacrylate glue (VetBond)</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>12612</td>
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	</tbody>
</table>

a large lateral craniotomy procedure for mesoscale wide-field optical imaging of brain activity

开颅是一种常用执行程序以暴露脑用于体内实验。在小鼠研究，大多数实验室利用小开颅，一般为3毫米×3毫米。此协议引入用于产生基本上更大7毫米×6mm的颅窗露出最一个大脑半球的在小鼠颞和顶叶皮层的方法（ 例如，囟2.5 - 4.5毫米，侧0 - 6毫米）。为了执行该手术，头必须倾斜大约30°，并且许多颞肌的必须缩回。由于大量的骨质去除，这种方法仅用于急性实验在整个手术和麻醉实验动物。 这种创新的大的横向颅窗口的主要优点是提供了皮质的两个内侧和外侧的区域同时访问。这个大单边颅窗口可用于研究细胞之间的神经动力学，以及通过组合多电极电生理记录，神经元活动（ 例如，内部或外部成像），和光遗传学刺激的成像作为不同的皮层区之间。此外，这种大开颅手术也暴露出皮层血管大面积的，允许横向皮质血管的直接操作。

<html>为了研究在单个半球内皮层区之间的相互作用，我们使用了跨越矢状窦和5延伸出的大的开颅 - 6毫米横向。此颅窗口包括初级（马达，体感，视觉，听觉），仲（电机，视觉）和关联（脾后，扣，顶叶协会）右脑半球（ 图3A）的皮层。对于这项工作，我们使用电压敏感染料（VSD）成像，这反映在膜电位3变化。该协议也将是其它外在的（ 例如 ，钙17和谷氨酸18成像）或内在成像实验是有用的。当刺激后肢，前肢，晶须，视觉的，或使用0.5％异氟烷轻度麻醉的小鼠的听觉系统，我们观察到皮质去极化的共识图案（&gt;图3B）。与以前的研究相一致5，19，20，21，22，我们发现C2桶皮质的短暂触觉刺激导致初级躯体感觉区的激活，以及功能相关的区域内的反应"孤岛"。例如，主运动皮层（M1）或躯体感觉皮质的二次表示（S2; 图3Bi）。单个1毫秒音调PIP（25千赫兹）刺激导致初级听觉皮层（A1）约20毫秒后的听觉刺激（ 图3Bii）的活化。在接下来的几毫秒的时间，去极化整个听觉皮层传播，并传递到邻近二次躯体感觉皮层。音开始后约25毫秒，次级皮层去极化会出现，位于1.07; 0.2毫米内侧和后部相对1.9±0.1mm至囟（N = 9只小鼠）。这大约是顶叶联合区（PTA）的位置。该VSD信号然后被传播到其他皮质关联区域位于包括脾后（RS）中线区和扣带皮层（CG）。因此，听觉刺激导致了两个独立的重点领域的激活，从VSD去极化传播的行波到中线皮质内更大的区域。用1毫秒的绿色对侧眼的焦点刺激LED脉冲，导致在40ms内（ 图3BV）初级视觉皮层的激活。视觉皮层的这种初级活化依次为:（1）空间扩张VSD去极化到位于内侧相邻区域，横向，和前到初始激活的区域; （2）第二内侧皮质区域的去极化约50ms刺激（N = 8个实验）位于沿SA后<html> gittal缝合。这是类似于前肢的感官刺激（ 图3Biii），后肢（ 图3Biv），C2晶须，或试听。从前肢，后肢或试镜的感官刺激诱发VSDI响应初始激活的各初级感觉皮层，随后活性的各向异性扩散，以及大约20个皮质中线激活 - 刺激后40毫秒。这个结果是相似的，从视觉和晶须刺激的反应。沿着这些中线路线和相同区域的频繁激活感觉诱发活性的自发活动6的传播可能表明这些区域是小鼠皮层的连接核心，其中感官信息可以与自发皮质活动整合的中心轮毂。 2fig1.jpg" /&gt; 图1。 手术设置和准备。 （A）小鼠头部剃毛，洁净，旋转横向曝光约30°，并与耳棒的钝端固定。异氟醚麻醉剂经由鼻部件和齿架递送。鼠标覆盖有自粘塑料包装以增加不育和温暖。 （B）特写示出皮肤和从壁层颅骨外板骨膜移除，颞肌是不变。 （C）将颞肌除去曝光时间板与鳞骨，注意浅静脉没有损坏。 （D）到固定之前，头板被定位到正确的位置用蜡。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52642/52642fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2。 一步一步的外科手术。 （A）的平板连接到在前面和后面的位置用乙酸乙酯氰基丙烯酸酯胶颅骨。注意前囟的位置（黑色片三角胶带的）。 （B）是通过将头板和颅骨之间加厚牙科用粘固剂制备的颅窗口。请注意，前囟门和鳞地标仍然可见。 （C）按照水泥的干燥，脑缓冲液加入到软化颅骨和防止硬脑膜的粘附。一种卷纸将有助于消除之前钻脑缓冲区。 （D）开颅手术的边缘已经取得。注意脉管系统更容易通过附近的牙科水泥边缘变薄的骨可见。 （E）的顶和颞颅骨板已经祛瘀ED和硬脑膜可见。注意从轻微出血硬脑膜，这是正常的血液污点。 2下仔细检查 - 放大4倍，就会发现血管两层，一个在硬膜和其他的软。 （F）硬脑膜被除去露出了原始皮质。软脑膜血管是鲜红的，没有瑕疵存在。需要注意的白色硬脑膜杂散件在颅窗口的边缘。在这个例子中，有在颅窗口，从而迅速凝结的后部轻微硬脑膜渗出。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52642/52642fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/52642/52642fig3.jpg" /> 图3。 在感官刺激的多种形式独特和共识激活模式。 &lt;<html> / A &gt; （A）单侧开颅手术示意图，显示成像的皮层区域。 （B）每张图像中白色圆圈表示的宽边单侧开颅手术的显微照片。 （i）刺激（刺激）对侧C2晶须，（ii）听觉刺激，（iii）对侧前肢刺激，（iv）对侧后肢刺激和（iv）对侧后肢刺激和（iv）对侧后肢刺激（v）用发光二极管（LED）对对侧视觉刺激。在主要感觉皮质激活后10 - 25 ms，所有形式的感觉刺激（白色箭头）都有中线激活。答案是20次试验的平均值。第二行（ii）左侧第二位的图像表示前（A），后（P），内侧（M）和横向（L）方向。经Mohajerani 等人授权， 2013年修改。p_upload / 52642 / 52642fig3large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity at JoVE.com

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

该协议提出了在小鼠大脑皮质的时间和顶叶区域产生大单方面开颅的方法。这对于实时成像在皮质半球的一个广阔的区域是特别有用的。

大脑侧向颅骨切开术大脑活动的中尺度广角光学成像

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

a-large-lateral-craniotomy-procedure-for-mesoscale-wide-field-optical

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.2K 次观看.  University of Lethbridge. 该协议提出了在小鼠大脑皮质的时间和顶叶区域产生大单方面开颅的方法。这对于实时成像在皮质半球的一个广阔的区域是特别有用的。 

视频: A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

依赖国家对TMS的:一看动机幻视行为

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

科研

神经科学

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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.

小鼠大脑皮层细胞组织的大尺度三维成像

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

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

动物实时功能磁共振成像大脑映射

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

斑马鱼游泳期间后横向线阿弗伦特神经元的活动

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

体内 使用大颅窗的小鼠宽场和双光子钙成像

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...