Name: optogenetic functional mri
Uploaded: 2016-04-19T13:00:00.000+00:00
Duration: 6 min 6 s
Description: Watch this Scientific Journal Video about Optogenetic Functional MRI at JoVE.com

This work was supported through funding from the NIH/NIBIB R00 Award (4R00EB008738), Okawa Foundation Research Grant Award, NIH Director's New Innovator Award (1DP2OD007265), the NSF CAREER Award (1056008), and the Alfred P. Sloan Foundation Research Fellowship. J.H.L. would like to thank Karl Deisseroth for providing the DNA plasmids used for the optogenetic experiments. The authors would also like to thank Andrew Weitz and Mankin Choy for editing the manuscript and all the Lee Lab members for their assistance with the ofMRI experiments.

伦理学声明:实验步骤这里已经批准了斯坦福大学的机构动物护理和使用委员会（IACUC）。 1.准备跳线和套圈植入注意:尽管接插电缆和套圈植入物是市售的，生产这些内部使专门设计和将花费更少。 <ol><li>用于在大脑从激光传送光到植入物制成纤维跳线，第一切割光纤到所需的长度。 <ol><li>用光纤切割刀，终止该光纤，以产生在终端点的扁平端。如果纤维已经涂有一外套，剥去护套了与预先纤维剥离工具。 </li><li>切割光纤以产生用于电缆的所需长度的另一端，确保电缆足够长以从光源延伸到动物的孔内扫描仪。 </li></ol></li><li>下一步前不久1比射到一块铝箔，作为环氧胶变混合后太粘使用5分钟:混合于1环氧胶少量。 </li><li>用一个小木棍，环氧树脂胶轻轻涂抹到将被放置在陶瓷插芯的凹侧内，然后申请一小滴上套圈的侧平的表面纤维的一部分。将其插入套箍以后，保证光纤（&lt;0.5mm）的小长度从陶瓷套圈的平面侧突出。允许环氧树脂胶硬化O / N以获得最佳效果。 </li><li>在光纤跳线的侧将连接到激光光源，轻轻环氧树脂胶适用于将被放置在FC / PC连接器的套圈的凹侧内的纤维的部分，然后应用一个小拖放到套圈的平面侧的表面。将其插入套箍以后，确保该纤维（&lt;0.5mm）的小长度从套圈的平坦侧突出。允许环氧树脂胶硬化O / N以获得最佳效果。 </li><li>通过使用镊子应用上套圈平缓向下的压力抛光套圈用于使用研磨盘的电缆两侧的平坦端，而从3微米至1微米至0.3微米的粒度使得在氧化铝上研磨板（图8转）。 </li><li>检查用放大100倍的显微镜套圈的平板终端。确保该平端，包括光纤表面本身的表面上，是自由的任何环氧树脂胶;继续研磨如果环氧树脂胶留在表面上。确保光纤表面没有破裂或碎裂。 注意:确保人员采取适当的激光安全培训课程和操作激光设备前穿激光防护镜。 </li><li>光纤跳线通过FC / PC连接器连接到激光光源和调整的FIBER尖耦合器的焦点。测量用功率计的光纤的光传输功率，以保证有足够的光输出。 注:以下步骤是制备陶瓷插芯与光纤慢性植入到大脑;陶瓷套圈是在中心中空和携带光纤到大脑内的贴片电缆传送光到感兴趣区域（ROI）的区域。 </li><li>用光纤切割刀，终止该光纤，以产生在终端点的扁平端。如果纤维已经涂有一外套，剥去护套了与预先纤维剥离工具。 </li><li>切割光纤的另一端，以产生用于植入所需的长度进入大脑。使用立体定向图谱定位大脑内的ROI确定光纤的长度。 <ol><li>例如:目标背侧海马在大鼠，低于前囟3.5微米，确保从ferrul突出的纤维长度Ë为3.5mm +0.25毫米，占颅骨厚度允许的误差幅度。因此，检查纤维的最终长度为3.5mm +0.25毫米10.5毫米（套圈的长度）=14.25毫米。 </li></ol></li><li>混合，在1环氧树脂胶少量:下一步前不久1比射到一块铝箔（环氧胶混合后成为使用5分钟太粘）。 </li><li>用一个小木棍，环氧树脂胶轻轻涂抹到将被放置在陶瓷插芯的凹侧内，然后申请一小滴上套圈的侧平的表面纤维的一部分。将其插入套箍以后，保证光纤（&lt;0.5mm）的小长度从陶瓷套圈的平面侧突出。允许环氧树脂胶硬化O / N以获得最佳效果。 </li><li>用抛光盘用的镊子适用于套圈温和的下行压力，同时使8字形旋转抛光套圈的平端S于氧化铝研磨片（从3微米至1微米至0.3微米的粒度）。 </li><li>检查用放大100倍的显微镜套圈的平板终端。确保该平端，包括光纤表面本身的表面上，是自由的任何环氧树脂胶;继续研磨如果环氧树脂胶留在表面上。确保光纤表面没有破裂或碎裂。 </li><li>耦合抛光套圈与套圈套筒光纤跳线和贴片电缆连接到一个激光光源。测量在用功率计的光纤的前端的光发射功率，以保证有足够的工作效率。 </li><li>保持一个记录从贴片电缆的套圈要求每个套圈植入到输出在光纤（2.5毫瓦在这个协议）的前端的所需功率电平的功率输出。丢弃套圈具有超过50％的衰减率，并具有非圆形的输出图案。 </li></ol> 2.立体定位小鬼lantation手术和注射病毒<ol><li>确保涉及使用动物的任何实验程序由本地IACUC批准。按照无菌程序，包括使用无菌手套，无菌口罩，无菌手术巾和消毒的手术器械维持在生存手术无菌条件。 注意:确保医生都穿着适当的个人防护装备（PPE），包括在开始操作之前，安全护目镜。与腺相关载体（AAV）时，注意避免飞溅遵循标准的生物安全程序。 AAV的垃圾在生物危害容器中进行处理。 </li><li>加载具有足够的AAV微升注射器注射到动物加额外考虑到潜在的体积损失（总每只动物4微升），保持注射器在冰上使用前。在这个协议中，用于在4×10 12 VG / ml的滴度AAV5-CaMKIIa-hChR2（H134R）-EYFP。将异氟醚下的动物È麻醉用感应室，连接到一个精密的异氟烷蒸发器组在3 - 4％与氧气源。 </li><li>用电动剃须刀刮胡子的头部和使用聚维酮碘和70％乙醇冲洗皮肤进行手术三倍擦洗。 </li><li>一旦动物在深麻醉（趾检查反射和呼吸频率），固定动物的头骨与内部听觉定位螺栓和齿杆立体定位仪。 注:整个过程将需要1 - 2小时，从麻醉诱导恢复。 </li><li>设置麻醉到适当的水平（1 - 在蒸发器3％异氟烷），并持续监控动物的生命体征，调整麻醉必要时保持40次呼吸/分钟的〜呼吸速率。管理上的动物的眼睛的眼用软膏，以防止干燥时在麻醉下。 </li><li>使15 - 20毫米中线切开头皮用解剖刀并使用外科止血缩回头皮ttached至骨膜。参考Lambda和前囟门钻头定位在对投资回报率的坐标。 </li><li>钻一个小的开颅手术 - 在用牙钻的投资回报率（2 3毫米），，注意不要刺破大脑。慢慢插入通过开颅附着到微升注射器在大脑中的ROI针。 </li><li>用微量泵控制器，注入2微升载体溶液进入投资回报率。使用150升/分钟的流速，以避免组织损伤。注射完成后，慢慢地去除注射器，以0.5mm / min的速度之前等待10分钟。 </li><li>注射之后，擦干头骨表面。参考拉姆达和前囟，以确认坐标，然后插入套圈植入到目标深度（例如:3.5毫米以下囟为背侧海马）在约0.5毫米/分钟的速率。安装套管植入物用牙科水泥颅骨。牙科水泥凝固后，密封缝合切口（SI泽5-0老鼠）周围的牙齿水泥盖。 </li><li>手术后，将它在笼中单独圈养，对麻醉复苏加热器的顶笼一半的动物。无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。不要将动物在该公司其他动物的，直到它已完全恢复。 </li><li>对于手术后的疼痛管理，辖丁丙诺啡皮下注射0.05毫克/千克24小时的剂量，每12小时。每日施用抗生素粉末在切口部位3天。 </li><li>手术后两周左右取出缝合线，以防止结痂。 </li><li>等待4 - 6周病毒注射光遗传学基因的表达足够进行实验前了。 </li></ol> 3.光遗传学功能磁共振成像注意:锻炼围绕核磁共振成像扫描仪的永久磁场谨慎。安全设备，用品耳鼻喉科，包括函数发生器，光源，呼吸机，二氧化碳监测仪和气体罐，足够远（至少超过5高斯限制）。 <ol><li>放置气体麻醉下的动物用感应室，连接到一个精密的异氟烷蒸发器组在5％与氧气源。 </li><li>一旦动物是在深度麻醉（检查趾反射和呼吸速率），根据在里瓦德等人详细描述的协议插管动物。（2006）由二氧化碳图26，以允许二氧化碳的监控。注:插管在成像过程中保持呼气的 CO 2的正常水平是至关重要的。 </li><li>确保动物在扫描仪摇篮。 注意:在这个协议中，托架被定制生产，但这样的支架也可商购。大鼠摇篮功能，以确保扫描仪内的动物交付麻醉，热空气和运动的限制。 </li><li>递送异氟烷的混合（rangin得自1.2 - 1.5％）的大约60％的一氧化二氮和40％的氧气通过管道通过的摇篮。确保动物的头被牢固地固定，以避免运动伪影。 </li><li>通过在支架管提供热空气，并插入直肠温度计润滑剂监测体温。管理上的动物的眼睛的眼用软膏，以防止干燥时在麻醉下。 </li><li>光纤跳线连接到激光光源并测量接线电缆的套圈用功率计尖端的输出。 </li><li>调整到适当的功率电平（在步骤1.15预先确定的），以产生在大脑内植入光导纤维的前端所需的输出（2.5毫瓦在此协议）。由于从光纤输出过大的功率可能会导致大脑内部组织损伤或引起，这将产生信号假象组织加热，不显著加大激光的功率输出超出预期的输出。 </li><li>从黑电带的锥形植入物防止漏光并覆盖该动物的眼睛。耦合光缆上有一箍套筒动物套圈植入物。 </li><li>放置线圈在动物的头。将动物摇篮到扫描仪的孔。 注意:在这个协议中，单环发送 - 接收线圈被定制生产和预调谐以接收从脑组织的最佳无线电频率。 </li><li>监控呼吸率和体温在此过程中，调整作为必要保持限度内生理值的人工换气装置和加热器（确保呼气的 CO 2是3 - 4％通过旋转上呼吸机的旋钮调节冲程频率和音量，并且温度是通过点击用于温度设定的箭头）37℃。 </li><li>选择一个定位序列图像动物头位置。如果将b雨不是在等中心，调节动物头位置，然后重复所述定位扫描直到大脑在等中心。选择线性匀场序列和点击的顺序选择窗口负荷。接下来，单击开始减少磁场的不均匀性。 注:匀场是会直接影响功能磁共振成像数据的完整性的关键一步。 </li><li>选择T2加权序列和点击的顺序选择窗口负荷。接下来，单击开始之前，fMRI来获得T2加权高分辨率日冕解剖图像检查对大脑的整体完整性，并确认光纤植入的位置。 注意:这些图像可以被用作解剖覆盖了ofMRI扫描系列。 </li><li>从MRI扫描仪的触发端口连接BNC电缆给函数发生器，使激光光源根据一个实验刺激范式驱动。 注:在这个协议中，刺激paradigm是30秒基线其次是20秒开/ 40秒关六分钟。 </li><li>选择多层梯度回波（GRE）序列，并单击顺序选择窗口负荷。接下来，单击开始收购平面35毫米×35毫米（2D FOV）冠状切片用0.5毫米×0.5毫米×0.5 MM空间分辨率。 注意:这里使用的GRE序列具有TR / TE的重复时间（TR）和回波时间（TE）=毫秒750/12和30°翻转角度。 </li><li>在扫描结束时，从扫描仪中删除的动物和监视，直到它已经从麻醉觉醒并能维持胸骨斜卧。 </li></ol> 4. ofMRI数据分析注意:如在高通量ofMRI 27的出版物描述在MATLAB执行以下步骤。 <ol><li>原始扫描数据已传输到用于分析的计算机后，使用滑动窗口重建更新每个TR的形象，四交错螺旋读出，750毫秒TR和12毫秒回波时间获取每次扫描系列23片。相反，其中只有毕竟交错采集每个片新功能磁共振图像重建传统的重建，滑动窗口重建方法获取每个中间层27后的重建图像。 </li><li>如先前27描述的处理用于两维网格重建，运动校正，以及时间序列的计算的原始数据。 </li><li>使用阈值的一致性方法，通过如前面描述的27计算每个相对于刺激前收集到的基准期体素的BOLD信号的调制率来确定激活体素。计算相干值作为傅立叶幅度变换在反复刺激循环通过所有频率成分8的求和的平方除以频率。 </li><li>使用的时间序列数据为各Voxel，平均运动修正的图像，属于同一刺激范例连续扫描第一。如前面所述27的平均4D图像然后对齐六学位的自由的刚体变换加上各向同性的缩放范围内与动物之间的比较常见的坐标系。 </li></ol>

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The authors have nothing to disclose.

成像期间的对象的运动是伪影，可导致数据损坏的一个显著源。适当地固定在成像摇篮动物可以尽量减少这种假象，这将保持适当的麻醉水平。这里，我们用异氟烷但替代麻醉药，如美托咪定或氯胺酮和赛拉嗪，也应考虑。然而，麻醉剂的水平和选择可以在大脑中影响很多参数，包括BOLD反应28。异氟醚可导致神经元兴奋29变化。其他麻醉剂也可以影响GABA突触抑制30。因此，当执行ofMRI鉴于其对影响神经元的活性的能力麻醉的选择是重要的。 ofMRI在没有麻醉的是可能的，但可以与从动物，可如果动物是习惯于减小增加运动被挑战;先前已经进行这样的清醒ofMRI一个研究次将避免对大脑9,10麻醉的混杂影响。后处理运动校正算法可用于大大减轻运动的影响。几个这些方法存在的，包括在这个协议中，其中下校正的基准图像的平方成本函数和图像的总和最小化所使用的逆高斯 - 牛顿算法。因为它实现了快速和强大的运动校正，采用了GPU并行平台设计，以减少处理时间27的算法是很有用的。 在这个协议数据重建，在MATLAB环境定制编写的软件被用于二维网格重建，其中，螺旋形的样品被重建在k-空间分为网格图像31-33。通过计算每个相对于现有刺激所收集的基线期体素的BOLD信号的调制率被产生的时间序列数据。素的时间序列进行顺chronized与0.35或更大的相干性值被定义为激活体素光遗传学刺激块;这种连贯性值对应于小于10-9的P值8。计算相干值作为傅立叶幅度变换在反复刺激循环通过所有频率分量8,27的求和的平方除以频率。可以使用Bonferroni校正多重比较来控制Familywise错误。分析的替代方法，可以使用包括参数统计测试如一般线性模型（GLMS）。相干方法需要比以往一般线性模型的HRF的少先验知识。因此，使用ofMRI探索数据时，它是有利的。然而，仅可用于与块设计数据的连贯性方法或选择事件相关的设计具有固定刺激间隔，并且可以不与其它事件RELA ofMRI数据被用于特德设计或混合设计。随后，动态因果模型（DCM）可用于分析通过ofMRI确定大脑区域之间的相互作用。 DCM是功能磁共振成像34时从系统响应实验输入功能连接的分析，制定了贝叶斯统计技术。 对于ofMRI其他技术问题都在这里讨论。植入物可被损坏或脱落，导致从研究中除去受影响的动物。再植入手术不推荐，因为针对同样的投资回报率在原来的植入手术，由于动物福利问题的更多不确定性。因为投入到每个动物对象的时间和资源的显著量，考虑到材料的强度是用于ofMRI研究使用选择合适的牙科用粘固剂时显著关注。植入手术是在最大化IMPLAN长寿的关键因素T和动物主题。例如，确保颅骨是施加牙科水泥和放置水泥的足够量围绕陶瓷套圈植入物可以保证在研究过程中在动物的电位月之久的时间表稳定性之前干燥。此外，替代的笼子设计可以探索和与当地动物护理设施讨论，以避免与导线顶部保持食物和水的笼子，往往伸入笼和用于动物损害植入物提供了机会。重要的是，牙科用水泥必须仔细选择，以减少影响成像和替代水泥可以通过应用在一个扫描器在动物实验中使用前被测试到幻象和成像伪影。试验和错误的各种牙科粘固表明，在此协议中使用的水泥给出相对较少的工件。在执行ofMRI另一技术挑战是光纤放置在预期的ROI的准确度，给出的抽emely小的距离，可以在大脑中35原子核之间存在。在完成植入手术后，T2加权解剖扫描可用于通过覆盖到脑图谱，以确定正确位置。外科医生和实践进行这些手术的技能可以提高正确的就业率。在预定的ROI的特异性和视蛋白的表达可以在研究结束时由灌注动物和固定脑，利用免疫组织化学或标记到视蛋白可视化记者蛋白的内源荧光进行验证。这些报告蛋白也可与其它蛋白质共定位，以确保视蛋白是在所需神经细胞类型1,8,15,25表达。如前面提到的，工件可以从光递送22由于组织加热执行ofMRI时出现。该组织加热导致的弛豫时间修改，造成假BOLD信号。为了确保交流ofMRI期间将光刺激导致释放等是不是由于此工件，视蛋白阴性对照应执行，其中与对照的荧光团的载体（如AAV-CaMKIIa-EYFP）注射盐水注射的动物或动物经受ofMRI。此外，只有构建良好的光纤的植入物具有良好的光传输效率，应使用以去除需要使用高的激光功率。 ofMRI研究已经在其中错误激活由于组织加热被执行尚未问题1,6-8,10,11。 关于载体引入所需光遗传学基因为神经元中表达的选择，是不知道自动增值服务，以引起人类疾病，因此一个方便的选择，给定的使用这些试剂（BSL-1）所需的较低的生物安全水平。此外，载体核的众多随身携带的股票，并与多种血清型不同的光遗传学基因打包自动增值服务。 AAV的血清型，必须故选BASED的预期细胞群的目标，以确保最佳的表达水平36,37。慢病毒也可使用，但要求较高的生物安全水平。对于光遗传学基因的足够表达所需的时间取决于所用，所用的特定的AAV和在具体的实验范例的具体的动物模型是可变的。在这个协议中，Sprague Dawley大鼠11周龄的使用和光遗传学研究病毒注射后开始四到六周。转基因小鼠也可以在光遗传学研究中使用。有必要执行导频实验，以确定为视蛋白的充分表达所需时间的特定量。刺激范例可以根据所使用的特定视蛋白而变化。在这个协议中，AAV5-CaMKIIa-hChR2（H134R）-EYFP的使用量和刺激模式是开/ 40秒关20秒。如果使用SSFO，刺激的模式将有所不同，因为SSFO只需要光的短暂脉冲以作交流tivated然后光的另一波长的简要脉冲终止。 执行ofMRI阻止从与光纤活动电缆套箍植入物界面的光泄漏光遗传学刺激期间，以防止视觉刺激始发混杂大脑信号，即使当动物被麻醉时附加的临界关注。黑电带的锥体可用于阻挡从套圈的光，并以覆盖该动物的眼睛。重要的是，包括受试者的呼气的 CO 2和体温生理值必须正确地保持整个成像持续时间。在37℃下4％和体温-呼气的 CO 2应该保持3之间。另外，匀场序列中的磁场开始ofMRI扫描大大确定所得BOLD数据的质量之前降低尽可能多的不均匀性越好。这些因素控制是生产可靠ofMRI数据的关键。在这个协议中，DPSS激光器被用作光源光遗传学刺激。由于激光是连贯的，超过足够的力量可以很容易地通过光纤提供。耦合到光纤的LED光源可得自商业供应商，但是具有光透射功率下降的缺点。激光光源确实需要对准到每一个特定的光纤活动电缆，但与实践，对准可以在几秒钟到几分钟内完成。 ofMRI的未来的应用包括使用下一代视蛋白如红移视蛋白，以使成像过程中无创性刺激的。此外，MRI兼容的脑电图或与光纤植入物沿着类似的记录电极的植入可以允许除了MRI的高空间分辨率数据采集高时间分辨率的数据。 ofMRI与electrophysiological记录可以提供关于大脑的功能连接的大量信息。总之，ofMRI的监测整个大脑响应于由遗传或解剖身份定义的特定细胞群的刺激动力使得ofMRI在神经性疾病的研究和健康脑的连接组学的使用的重要工具。

光遗传学功能磁共振成像（ofMRI）是一种新技术，它结合高场磁共振成像的空间分辨率与光遗传学刺激1-11,38的精度，从而使在整个官能神经回路的细胞类型特异性的映射和它们的动态脑。光遗传学允许通过引入感光跨膜电导通道，称为视蛋白被靶向用于刺激的特定细胞类型。神经回路的具体内容是遗传修饰以表达这些渠道，扶持活动的毫秒时间尺度调制的完整的大脑1-15。磁共振成像提供通过血氧水平依赖（BOLD）的信号16-18，其中提供的神经元活动的间接测量的测量确定对特定的神经回路的光遗传学刺激大脑的全局动态响应的非侵入性的方法。 这两种技术的结合，称为光遗传学功能磁共振成像（ofMRI），是在刺激过程中记录的大脑活动的其他方法，如电有利的，因为它可以提供在相对较高的空间分辨率在整个大脑的图。这使得能够响应于定向刺激从刺激部位很远的距离，而不需要侵入记录电极1-11中植入的神经元活动的检测。 ofMRI超过fMRI的期间执行电刺激的更传统的方法，该方法可以招募不同类型的细胞在电极附近，从而挫败每个群体19的因果影响是有利的。另外，用于电刺激的电极和所产生的电流可以MR成象20中产生伪影。事实上，ofMRI使全球大脑活动的影响从具体modulati观察上的通过使用先进的基因定位技术，如Cre的lox系统在转基因动物或使用启动子的多种细胞类型。与全脑监测组合光控制是可能的ofMRI通过使用两个NpHR的抑制和的ChR2激发特定的细胞类型。可用于ofMRI利用光遗传学工具包也迅速随着时间的推移提高引进视蛋白的增加光敏感的或改进的动力学，稳定阶跃函数视蛋白（SSFOs）或红移视蛋白的可能否定了植入式光纤的要求光学，成像21期间启用无创性刺激。这些可能性是不具备的电刺激。 然而，从组织加热所得由于大脑中的光递送信号工件已经报道22，其中弛豫时间温度引起的变形例已被示出，以产生假性做激活。因此，在执行ofMRI研究人员应该意识到这种潜在的变乱的。通过适当的设置和控制，这个问题可以得到解决。此外，测量在fMRI的血流动力学响应的相对低的时间分辨率可以是用于该技术的某些应用中的限制因素。 这个协议首先描述了光纤植入，使特定波长的光的递送深入到体内的脑的结构。该协议随后介绍了立体定向手术精确的大脑区域交付视蛋白编码病毒载体。接下来的协议描述同时光刺激时全脑功能磁共振成像的过程。最后，协议概括了获得的数据的基本数据的分析。 值得注意的是，这里所描述的光遗传学需要光递送慢性植入物。然而，光纤的植入物是稳定的，生物兼容，从而允许纵向扫描和在数月23,24的神经电路的调查。 总之，准确的刺激和ofMRI的全脑监测能力是在使ofMRI为脑的连接组学的研究的强有力的工具的关键因素。另外，它可以提供新颖的洞察当与不同的动物模型耦合的神经系统疾病25的机制。的确，ofMRI已用来阐明发作8相关联的不同的海马分区域的网络活动。因此，有志于应答系统级神经科学实验室的问题会发现的重要性这种技术。

<table><tbody><tr><td>7 Tesla scanner</td><td>Agilent Technologies</td><td>Discovery MR901 System</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Charles River</td><td>Crl:SD</td><td>11 weeks old</td></tr><tr><td>fiber cleaver</td><td>Fujikura</td><td>CT-05</td><td></td></tr><tr><td>multimode optical fiber</td><td>Thor Labs</td><td>AFS105/125Y</td><td></td></tr><tr><td>fiber stripper&amp;nbsp;&amp;nbsp;</td><td>Thor Labs</td><td>T08S13</td><td></td></tr><tr><td>ceramic split sleeve</td><td>Precision Fiber Products</td><td>SM-CS1140S</td><td></td></tr><tr><td>epoxy glue</td><td>Thor Labs</td><td>G14250</td><td></td></tr><tr><td>cotton-tipped applicators</td><td>Stoelting Co.</td><td>50975</td><td></td></tr><tr><td>multimode ceramic zirconia ferrules</td><td>Precision Fiber Products</td><td>MM-FER2002</td><td></td></tr><tr><td>FC/PC multimode connector</td><td>Thor Labs</td><td>30128C3</td><td></td></tr><tr><td>fiber optic polishing disk</td><td>Precision Fiber Products</td><td>M1-80754</td><td></td></tr><tr><td>aluminum oxide lapping sheet, 0.3 &amp;micro;m</td><td>Thor Labs</td><td>LFG03P</td><td></td></tr><tr><td>aluminum oxide lapping sheet, 1 &amp;micro;m</td><td>Thor Labs</td><td>LFG1P</td><td></td></tr><tr><td>aluminum oxide lapping sheet, 3 &amp;micro;m</td><td>Thor Labs</td><td>LFG3P</td><td></td></tr><tr><td>binocular biological microscope 40X-1,000X</td><td>Amscope</td><td>B100</td><td></td></tr><tr><td>laser safety glasses</td><td>Kentek</td><td>KXL-62W01</td><td></td></tr><tr><td>473 nm DPSS laser</td><td>Laserglow</td><td>LRS-0473</td><td></td></tr><tr><td>594 nm DPSS laser</td><td>Laserglow</td><td>LRS-0594</td><td></td></tr><tr><td>Allen hex wrench set</td><td></td><td></td><td>2.0 mm (5/64&quot;) for alignment of fiber tip to focal point of coupler in the laser</td></tr><tr><td>power meter, Si Sensor, 400-1,100 nm</td><td>Thor Labs</td><td>PM121D</td><td></td></tr><tr><td>Isoflurane (Isothesia)</td><td>Henry Schein&amp;nbsp;</td><td>50033</td><td></td></tr><tr><td>isoflurane vaporizer with induction chamber</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>NanoFil 100 &amp;micro;l syringe</td><td>World Precision Instruments</td><td>NANOFIL-100</td><td></td></tr><tr><td>UltraMicroPump with SYS-Micro4 Controller</td><td>World Precision Instruments</td><td>UMP3-1</td><td></td></tr><tr><td>function generator</td><td>A.M.P.I.&amp;nbsp;</td><td>Master-8</td><td></td></tr><tr><td>small animal stereotax</td><td>David Kopf Instruments</td><td>Model 940&amp;nbsp;</td><td></td></tr><tr><td>Model 683 small animal ventilator&amp;nbsp;</td><td>Harvard Apparatus</td><td>550000</td><td></td></tr><tr><td>Type 340 capnograph&amp;nbsp;</td><td>Harvard Apparatus</td><td>733809</td><td></td></tr><tr><td>dental drill (rotary micromotor kit)</td><td>Foredom Electric Co.</td><td>K.1070</td><td></td></tr><tr><td>ophthalmic ointment (Artificial Tears)</td><td>Rugby</td><td>00536-6550-91</td><td></td></tr><tr><td>instrument sterilizer</td><td>CellPoint Scientific</td><td>Germinator 500</td><td>glass bead sterilizer</td></tr><tr><td>antibiotic powder</td><td>Pfizer</td><td>NEO-PREDEF</td><td>neomycin sulfate, isoflupredone acetate and tetracaine hydrochloride</td></tr><tr><td>buprenorphine painkiller</td><td>Hospira</td><td>NDC:0409-2012</td><td>schedule III controlled substance, 0.3 mg/ml stock</td></tr></tbody></table>

optogenetic functional mri

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

<html> 图1和图2示出了从20赫兹（15毫秒脉冲宽度，473纳米，30％占空比）的运动皮层的光遗传学刺激所得的代表性数据。基线30秒的刺激范式其次是20秒开/ 40秒关闭被使用了六个分钟。以前的研究已经表明，这种范例产生从光遗传学刺激1,8-健壮的BOLD信号。 图1示出了检测到的无论是在刺激（运动皮层）的本地站点激活体素和在丘脑，作为长范围突触连接的结果这些区域之间。 图2示出的时间信息可以从HRFS中收集，如丘脑响应延迟相比光遗传学刺激后运动皮层响应（低级初始斜率）。 p_upload / 53346 / 53346fig1.jpg"/&gt; 图1. 激活地图的BOLD信号的通过的CaMKIIa表达在运动皮质细胞光遗传学刺激诱 ​​发。活性体素，确定为那些显著同步反复刺激的相干值，示出覆盖在一个T2加权冠状解剖切片。数据收集了六分钟，30秒的时间（20秒的最初30秒基线和六个刺激周期上/ 40与473纳米的光，20赫兹，15毫秒脉冲宽度秒关闭）被浓缩成一个激活的地图。连续片相隔0.5mm至光纤植入的位置由三角形表示。 <a href="https://www.jove.com/files/ftp_upload/53346/53346fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53346/53346fig2.jpg" /> <s仲&gt;图2.血流动力学响应函数（左）相对于基线的BOLD信号的调制百分比是运动皮层（20秒开/ 40秒熄灭六刺激周期的刺激光遗传学期间显示在运动皮层和丘脑体素活跃与473纳米的光，20赫兹，15毫秒脉冲宽度）。灰色阴影误差棒表示ROI内跨激活体素的标准误差。 （右）时均响应由血流动力学响应函数（HRF）给出。丘脑HRF显示延迟反应相对激电机皮质。蓝条表示473纳米的光刺激的时期。灰色阴影误差线表示在六个周期的标准错误。 <a href="https://www.jove.com/files/ftp_upload/53346/53346fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Optogenetic Functional MRI at JoVE.com

Optogenetic Functional MRI

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

光遗传学功能磁共振成像

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Research

JoVE Journal

Neuroscience

14.7K 次观看.  Stanford University. This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

视频: Optogenetic Functional MRI

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

功能磁共振成像（fMRI）技术与听觉刺激鸣禽

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

科研

行为学

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

神经活性的光遗传学操作，调节自由移动小鼠的行为

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

推进视觉皮层映射以增强对视力障碍的评估

Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation

High-resolution retinotopic blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) with a wide-view presentation can be used to functionally map the peripheral and central visual cortex. This method for measuring functional changes of the visual brain allows for functional mapping of the occipital lobe, stimulating &gt;100&#176; (&plusmn;50&#176;) or more of the visual field, compared to standard fMRI visual presentation setups which usually cover &lt;30&#176; of the visual field. A simple wide-view stimulation system for BOLD fMRI can be set up using common MR-compatible projectors by placing a large mirror or screen close to the subject's face and using only the posterior half of a standard head coil to provide a wide-viewing angle without obstructing their vision. The wide-view retinotopic fMRI map can then be imaged using various retinotopic stimulation paradigms, and the data can be analyzed to determine the functional activity of visual cortical regions corresponding to central and peripheral vision. This method provides a practical, easy-to-implement visual presentation system that can be used to evaluate changes in the peripheral and central visual cortex due to eye diseases such as glaucoma and the vision loss that may accompany them.

作者聚焦:通过宽视角 fMRI 映射洞察视觉皮层研究 

We have developed techniques for mapping the visual cortex function utilizing more of the visual field than is commonly used. This approach has the potential to enhance the evaluation of vision disorders and eye diseases.

具有宽视网膜刺激的视觉皮层功能性磁共振成像 （fMRI）

High-resolution retinotopic blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) with a wide-view presentation can be ...

光遗传学功能磁共振成像

摘要

探索更多视频

此视频中的章节

功能磁共振成像（fMRI）技术与听觉刺激鸣禽

高分辨率人类中脑功能磁共振成像方法

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Optogenetically有针对性的前脑电路的激光扫描光刺激

神经活性的光遗传学操作，调节自由移动小鼠的行为

深部脑刺激与同步功能磁共振成像在鼠害

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

具有宽视网膜刺激的视觉皮层功能性磁共振成像（fMRI）

具有宽视网膜刺激的视觉皮层功能性磁共振成像（fMRI）

具有宽视网膜刺激的视觉皮层功能性磁共振成像（fMRI）

光遗传学功能磁共振成像

摘要

探索更多视频

此视频中的章节

功能磁共振成像（fMRI）技术与听觉刺激鸣禽

高分辨率人类中脑功能磁共振成像方法

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Optogenetically有针对性的前脑电路的激光扫描光刺激

神经活性的光遗传学操作，调节自由移动小鼠的行为

深部脑刺激与同步功能磁共振成像在鼠害

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

具有宽视网膜刺激的视觉皮层功能性磁共振成像 （fMRI）

具有宽视网膜刺激的视觉皮层功能性磁共振成像 （fMRI）

具有宽视网膜刺激的视觉皮层功能性磁共振成像 （fMRI）

具有宽视网膜刺激的视觉皮层功能性磁共振成像（fMRI）

具有宽视网膜刺激的视觉皮层功能性磁共振成像（fMRI）

具有宽视网膜刺激的视觉皮层功能性磁共振成像（fMRI）