我们感谢陈D.博士和颜C.博士分享AFNI脚本，为PV 5设置实时fMRI，并感谢AFNI团队提供软件支持。这项研究得到了NIH脑计划资助（RF1NS113278-01，R01 MH111438-01）和S10仪器资助（S10 RR023009-01）的支持给Martinos中心，德国研究基金会（DFG）Yu215 / 3-1，BMBF 01GQ1702，以及马克斯普朗克学会的内部资助。

这项研究是根据德国动物福利法（TierSchG）和动物福利实验动物条例（TierSchVersV）进行的。此处描述的实验方案由伦理委员会（§15 TierSchG）审查，并由国家当局批准（Regierungspräsidium，图宾根，巴登 - 符腾堡州，德国）。
1. 为小动物研究准备BOLD-fMRI实验装置
<ol><li>打开控制台软件以控制成像参数并获取MRI数据。 注意:建议的实时功能磁共振成像设置是利用控制台软件（版本6）的宏功能与AFNI的图像处理功能并行实现的。</li>
	<li>使用工作区资源管理器查找 MR 序列（即位置、定位器、弛豫增强快速采集 （RARE） 和 3D 回波平面成像 （EPI），然后将它们拖动并附加到扫描列表中。 注意:位置和定位器序列用于识别大脑中的感兴趣区域（ROI）。RARE序列用于解剖扫描。3D EPI 序列用于测量动态粗体响应。</li>
	<li>将预定义的宏脚本"Setup_rt3DEPI"和"Feed2AFNI_rt3DEPI"放在宏脚本路径中（例如，"/opt/（PV version）/prog/curdir/（用户名）/ParaVision/macros"）。激活"数据重建"用户界面菜单中的3D EPI重建选项，"图像前系列活动"和"执行宏"，然后在单击"扫描"按钮之前链接预定义的宏脚本"Setup_rt3DEPI"。 注意:宏脚本包含在 补充文件中。</li>
	<li>安装 AFNI 软件以进行实时 BOLD-fMRI 分析和可视化。</li>
</ol>2. 导尿通气手术
<ol><li>设置呼吸机和生理状态监测系统，如温度计、血压和呼吸记录，如图 1所示。使用呼吸机设置60±1呼吸/分钟的恒定频率，并使用带有反馈控制装置的MR兼容加热垫设置37°C的温度。</li>
	<li>在含有5%异氟醚的腔室中麻醉成年雄性Sprague-Dawley大鼠（300-600g）进行诱导，并从蒸发器提供2-2.5%异氟烷用于手术。通过捏住后爪并确认缺乏戒断反应来检查麻醉深度。</li>
	<li>用14G塑料插管插管动物进行通气（60±1次呼吸/分钟，70%空气和30%氧气的混合物）。将潮气末二氧化碳 （CO 2） 调整为 25 ± 5 mmHg29 范围内。 注意:插管对于通过功能磁共振成像实验维持适当的CO2 水平至关重要。</li>
	<li>将动物仰卧放在手术台上，并用电动剃须刀剃大腿。然后，用手术剪刀在剃光的皮肤上切开一个切口。 注意:切口的长度在纵向上约为1-2厘米。</li>
	<li>在切开区域下找到股动脉和静脉进行导管插入，并将单个股动脉和静脉与周围组织分开。</li>
	<li>用手术缝合线固定分离的股动脉的一侧，并用微型斗牛犬镊子固定另一侧。然后，在股动脉上的连接区域之间做一个小切口。</li>
	<li>通过小切口将导管插入股动脉，并用手术缝合线将导管和动脉绑在一起。使用生理监测系统持续监测动脉血压，使其在 80-120 mmHg 范围内，并定期测量动脉血气，以在扫描过程中保持最低 90 mmHg 的 pO 2 和 30-45 mmHg 的 pCO2。 注意:这种导尿对于在功能磁共振成像实验期间监测动脉血压至关重要。</li>
	<li>用丝编织手术缝合线固定股静脉的两端。然后，在股静脉的绑扎区域之间做一个小切口。使用镊子进行缝合。 注意:缝合线的大小约为1-2厘米。</li>
	<li>将导管插入股静脉。用手术缝合线将导管和静脉绑在一起。 注意:这种导管插入对于通过静脉施用α-氯蔗糖和在fMRI实验期间调整麻醉水平至关重要。如果动物没有很好地麻醉，它会开始自主呼吸。在这种情况下，必须给予更多的α-氯蔗糖以避免呼吸运动伪影。</li>
	<li>在剃光的皮肤上缝合手术切口。一旦外科手术完成，通过连接到股静脉的导管输注剂量为~80mg / kg的α-氯蔗糖推注，保持动物麻醉，并同时停止异氟醚给药。</li>
</ol>3. 将动物放入 MRI 扫描仪内
<ol><li>完成2.10步骤后，立即将麻醉动物转移到MRI扫描仪上，并将其固定在定制的摇篮上。</li>
	<li>在动物身上插入实时反馈直肠温度计以监测动物的温度。在动物的躯干下放置一个加热垫以控制温度。在MRI扫描期间将体温保持在37.0±0.5°C。</li>
	<li>在肌肉松弛剂泮库溴铵（~2 mg/kg/h）的混合物中连续递送具有~25mg / kg / h溶液的α-氯蔗糖，同时保持动物麻醉并减少fMRI图像中的运动伪影。根据生理状态调整药物量和通气速率，监测血压和呼吸。</li>
	<li>在动物的眼睛上施用眼药膏，以防止在fMRI实验期间干燥。用两个耳杆安全地固定动物的头部，以避免头部运动伪影。</li>
	<li>将收发器表面线圈固定在磁头上。在 MRI 测量之前，将线圈调谐并匹配到头部的拉莫尔频率（例如，14.1 T 上的 599 MHz）。 注意:在这里，直径为22毫米的线圈用于覆盖大鼠的整个大脑。</li>
	<li>将一对针状电极插入前爪皮肤中的手指 1 和 4 之间，并用手术胶带固定它们。然后，确认在将刺激输入电缆连接到这些电极30之后刺激工作正常。</li>
	<li>将动物插入MRI孔中，并将其大约放置在等中心。</li>
</ol>4. 测量解剖学磁共振成像
<ol><li>单击主用户界面中的校准菜单按钮。单击 调整平台 用户界面中的以下项目（请参阅控制台软件中的 "帮助 "菜单）执行MRI系统的校准:查找基本谐振频率，校准RF脉冲功率，设置最佳接收器增益，测量动物中的B0图以进行匀场，基于非局部自由感应衰减（FID）积分运行全局线性匀场。 注意:此步骤不到 2 分钟。</li>
	<li>通过单击"扫描"按钮运行位置序列，以找到MRI孔内动物的头部位置。如果磁头不在等中心，请在来回移动底座的同时调整磁头位置，直到磁头位于等心处。</li>
	<li>通过单击"扫描"按钮运行本地化器序列，以识别头部的投资回报率。选择映射填充程序并定义填充程序体积的 ROI 以覆盖定位器图像中的整个大脑，然后使用"填充程序最多"选项运行高阶（例如，2阶或 3 阶）匀场，以减少 ROI 处的主磁场 （B0） 不均匀性。 注意:当使用EPI序列时，高阶匀场是提高BOLD-fMRI数据质量的关键步骤。</li>
	<li>通过单击"扫描"按钮运行 T2 加权 RARE 序列，以获取冠状视图中覆盖整个大脑的解剖图像（例如，使用以下序列参数:重复时间 （TR） 4000 ms，有效回波时间 （TE） 36.1 ms，矩阵 128 x 128，视野 （FOV） 19.2x19.2 mm2、切片数 32、切片厚度 0.3 mm， 稀有系数 8）。 注意:在以下实时fMRI可视化步骤中，解剖图像用于将3D EPI图像注册为模板。</li>
</ol>5. 实时功能磁共振成像软件设置和功能磁共振成像响应可视化
<ol><li>打开终端窗口并使用以下命令转到实时 AFNI 插件路径: cd /home/（用户名）/rt_afni 注意:AFNI 插件脚本"afni_rt"包含在 补充文件中。</li>
	<li>使用以下命令和选项使用实时插件执行 AFNI 软件。 AFNI -RT -yestplugouts -DAFNI_REALTIME_MP_HOST_PORT=本地主机:（端口号） -DAFNI_REALTIME_Graph=实时 -DAFNI_FIM_IDEAL=（范式） 注意:在第一种情况下，代码允许外部程序与 AFNI 交换数据，而在第二种情况下，实时插件将尝试打开用户定义的本地主机和端口的 TCP 套接字。在第三种和第四种情况下，当获取实时功能磁共振成像数据时，代码将实时绘制功能磁共振成像数据的时间过程，并分别绘制用户定义范式在功能磁共振成像时间过程中的时间过程。有关更多详细信息，请查看 https://afni.nimh.nih.gov/pub/dist/doc/program_help/README.environment.html。</li>
	<li>监视使用命令"Dimon"定义的即将发布的 AFNI BRIK 文件，如图 2 所示，具有以下选项: Dimon -tr （TR of EPI） -nt （NRepetitions of EPI） -RT -退出 -infile_pattern实时*。布里克 -file_type AFNI 注意:"Dimon"是使用以下选项监视AFNI图像文件的实时采集的命令:"-rt"执行实时插件和"-infile_pattern（数据名称）。BRIK -file_type AFNI"，它允许插件读取特定的BRIK文件并将其发送到AFNI进行显示和格式化。有关更多详细信息，请查看 https://afni.nimh.nih.gov/pub/dist/doc/program_help/Dimon.html。</li>
	<li>使用带有以下选项的"pvcmd"命令: pvcmd -a JMacroManager JMMExecuteMacro -category $USER -macro Feed2AFNI_rt3DEPI 注意:此代码存在于宏脚本"Setup_rt3DEPI"中，用于在单击"扫描"按钮进行EPI采集后立即运行后台宏脚本"Feed2AFNI_rt3DEPI"。</li>
	<li>使用带有以下选项的"exec pvcmd"命令获取 EPI 采集参数。 exec pvcmd -a ParxServer -r ParamGetValue -psid $ParSpaceId -param （PVM parameters of EPI） -id 10 -args $AcqKey $ParSpaceId $ProcnoPath</li>
	<li>使用带有以下选项的"exec to3d"命令，在后台宏脚本"Feed2AFNI_rt3DEPI"中实时将EPI原始数据转换为AFNI文件。 exec to3d -omri -xFOV $FOV_X -yFOV $FOV_Y -zFOV $FOV_Z -prefix $LastVolName $ImgFormat$Path2dseq</li>
	<li>确保EPI几何信息与解剖方向一致。 注意:"to3d"AFNI 命令将自动运行，其中包含视场 （FOV） 和矩阵大小等几何信息，以将 fMRI 原始数据转换为一个 AFNI BRIK 数据，只要每个 TR 之后存储每个 3D 体积数据，如图 2 所示。可以使用"to3d"的几何信息参数更改图像方向。有关更多详细信息，请查看 https://afni.nimh.nih.gov/pub/dist/doc/program_help/to3d.html。</li>
	<li>打开电刺激隔离器并使用刺激块进行电前爪刺激，以进行一个诱发的 fMRI 研究（例如，3Hz，4s 脉冲宽度 300us，2.5mA）。 注意:在这里，模块设计范式包括 10 次刺激前扫描、3 次刺激扫描和 12 次刺激间扫描（每个时期 15 次扫描）。</li>
	<li>通过单击BOLD-fMRI研究的"扫描"按钮运行T2*加权3D EPI序列（例如，使用以下参数:TR / TE 1500 / 14 ms，矩阵64 x 64 x 32，FOV 19.2 x 19.2 x 9.6 mm 3和分辨率300 x 300 x 300 μm3）。 注意:单击"扫描"按钮后，将使用预定义的宏脚本实时监视和处理原始数据。转换一个 AFNI BRIK 数据集后，3D EPI 图像的体素时程图将显示在 AFNI 软件中，并针对每个 TR 自动更新。</li>
	<li>要将 EPI 图像叠加在解剖学 RARE 图像之上，请使用步骤 5.6 中的命令"to3d"将 RARE 图像转换为 AFNI BRIK 数据集，然后使用以下选项使用"align_epi_anat.py"AFNI 脚本将 EPI 图像注册到解剖图像: align_epi_anat.py -anat anatomy_template_al+orig -epi epi.$（epi data number）+orig -epi_base 1 -后缀 _volreg -rat_align -成本 LPA -epi2anat 注意:有关更多详细信息，请查看 https://afni.nimh.nih.gov/pub/dist/doc/program_help/ align_epi_anat.py.html。</li>
	<li>要处理粗体响应的功能图，请使用具有以下选项的"3dDeconvolve"命令计算具有特定刺激时间序列的 3D+time 数据集的反卷积: 3d去卷积 -输入 （输入文件名）+原始 -nfirst 0 -polort 3 -num_stimts 1 -stim_times 1 （刺激范式文件名） 'BLOCK（4，1）' -stim_label 1 前爪 -tout -fout -rout 注意:空间平滑或时间过滤等图像处理步骤已合并到自定义的 AFNI 数据处理脚本中。有关更多详细信息，请查看 https://afni.nimh.nih.gov/afni/doc/help/3dDeconvolve.html。</li>
	<li>要可视化粗体信号的功能图，请使用 AFNI 软件中的交互式聚类。打开"定义叠加"选项，然后使用 AFNI 用户界面菜单中的"集群"功能。</li>
	<li>最后一次fMRI扫描后，将动物从MRI扫描仪中取出，并根据批准的方案对其进行安乐死。 注意:AFNI的图像处理功能和最新控制台软件中的宏功能用于处理实时fMRI数据。宏功能的详细信息和说明可以从控制台软件的帮助菜单中找到。AFNI软件是一个免费软件，可以通过NIMH-AFNI网站直接下载。附上了用于在 AFNI 和控制台系统之间构建链接的相关脚本。</li>
</ol>

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Q., Ress, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+method+for+retrospective+correction+of+physiological+motion+effects+in+fMRI:+RETROICOR.">Image-based method for retrospective correction of physiological motion effects in fMRI: RETROICOR.</a> Magnetic Resonance in Medicine. 44 (1), 162-167 (2000).</li><li>Chang, C., Cunningham, J. P., Glover, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+heart+rate+on+the+BOLD+signal:+The+cardiac+response+function.">Influence of heart rate on the BOLD signal: The cardiac response function.</a> Neuroimage. 44 (3), 857-869 (2009).</li><li>Birn, R. M., Diamond, J. B., Smith, M. A., Bandettini, P. 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B., 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=Real-time+animal+functional+magnetic+resonance+imaging+and+its+application+to+neurophamacological+studies.">Real-time animal functional magnetic resonance imaging and its application to neurophamacological studies.</a> Magnetic Resonance Imaging. 26 (9), 1266-1272 (2008).</li><li>Cox, R. W., Jesmanowicz, A., Hyde, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+functional+magnetic+resonance+imaging.">Real-time functional magnetic resonance imaging.</a> Magnetic Resonance Medicine. 33 (2), 230-236 (1995).</li><li>Lee, C. C., Jack, C. R., Rossman, P. J., Riederer, S. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+functional+magnetic+resonance+imaging.">Real-time functional magnetic resonance imaging.</a> Methods. 25 (2), 201-220 (2001).</li><li>Posse, S., 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+new+approach+to+measure+single-event+related+brain+activity+using+real-time+fMRI:+Feasibility+of+sensory,+motor,+and+higher+cognitive+tasks.">A new approach to measure single-event related brain activity using real-time fMRI: Feasibility of sensory, motor, and higher cognitive tasks.</a> Human Brain Mapping. 12 (1), 25-41 (2001).</li><li>Decharms, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reading+and+controlling+human+brain+activation+using+real-time+functional+magnetic+resonance+imaging.">Reading and controlling human brain activation using real-time functional magnetic resonance imaging.</a> Trends in Cognitive Sciences. 11 (11), 473-481 (2007).</li><li>Bruhl, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+Sense+of+Real-Time+Functional+Magnetic+Resonance+Imaging+(rtfMRI)+and+rtfMRI+Neurofeedback.">Making Sense of Real-Time Functional Magnetic Resonance Imaging (rtfMRI) and rtfMRI Neurofeedback.</a> International Journal of Neuropsychopharmacology. 18 (6), (2015).</li><li>Cox, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFNI:+Software+for+analysis+and+visualization+of+functional+magnetic+resonance+neuroimages.">AFNI: Software for analysis and visualization of functional magnetic resonance neuroimages.</a> Computers and Biomedical Research. 29 (3), 162-173 (1996).</li><li>Liou, W. W., Goshgarian, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+the+effect+of+chronic+phrenicotomy+on+the+induction+of+the+crossed+phrenic+phenomenon.">Quantitative assessment of the effect of chronic phrenicotomy on the induction of the crossed phrenic phenomenon.</a> Experimental Neurology. 127 (1), 145-153 (1994).</li><li>Shih, Y. 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M., 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=Anesthesia+differentially+modulates+neuronal+and+vascular+contributions+to+the+BOLD+signal.">Anesthesia differentially modulates neuronal and vascular contributions to the BOLD signal.</a> Neuroimage. 195, 89-103 (2019).</li><li>Wu, T. 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=Effects+of+isoflurane+anesthesia+on+resting-state+fMRI+signals+and+functional+connectivity+within+primary+somatosensory+cortex+of+monkeys.">Effects of isoflurane anesthesia on resting-state fMRI signals and functional connectivity within primary somatosensory cortex of monkeys.</a> Brain and Behavior. 6 (12), 00591 (2016).</li><li>Liu, X., Zhu, X. 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Sascha Köhler是Bruker BioSpin MRI GmbH的员工。

功能磁共振成像信号的实时监测有助于实验者调整动物的生理机能，以优化功能映射。清醒动物的运动伪影以及麻醉作用是介导fMRI信号变异性的主要因素，混淆了信号本身的生物学解释31，32，33，34，35，36，37，38...

功能磁共振成像（fMRI）是一种非侵入性方法，用于测量与大脑神经活动相关的血流动力学反应1，2，3，4，5，6，7，8，9，例如血氧水平依赖性（BOLD），脑血容量和流量信号。在动物研究中，血流动力学信号可受到麻醉10、清醒动物11的应激水平以及潜在的非生理伪影（例如心脏搏动和呼吸运动12、13、14、15）的影响。尽管已经开发了许多后处理方法来为任务相关和静息状态功能动力学和连接映射提供fMRI信号的回顾性分析16，17，18，19，但很少有技术可以提供实时脑功能映射解决方案和动物大脑中的瞬时读数20（其中大部分主要用于人脑映射21， 22，23，24，25，26，27）。特别是这种实时功能磁共振成像成像方法在动物研究中是缺乏的。有必要建立一个功能磁共振成像平台，以实现实时脑状态依赖性生理阶段的研究，并为动物脑功能研究提供实时生物反馈刺激范式。
在本工作中，我们说明了使用MRI控制台软件的定制宏功能的实时fMRI实验设置，展示了对麻醉大鼠初级前爪体感皮层（FP-S1）中诱发的BOLD-fMRI反应的实时监测。这种实时设置允许使用现有的神经图像分析软件功能神经图像分析（AFNI）28，在功能图中可视化正在进行的大脑激活，以及以体素方式显示单个时间过程。用于动物研究的实时fMRI实验装置的制备在协议中描述。除了动物设置外，我们还提供详细的程序，使用最新的控制台软件和图像处理脚本来设置实时fMRI信号的可视化和分析。总之，拟议的用于动物研究的实时功能磁共振成像设置是使用MRI控制台系统监测动物大脑中动态功能磁共振成像信号的有力工具。

<table><tbody><tr><td>14.1T Bruker MRI system</td><td>Bruker BioSpin MRI GmbH</td><td>N/A</td><td></td></tr><tr><td>A365 Stimulus Isolator</td><td>World Precision Instruments</td><td>N/A</td><td></td></tr><tr><td>AcqKnowledge Software</td><td>Biopac</td><td>RRID:SCR_014279, http://www.biopac.com/product/acqknowledge-software/</td><td></td></tr><tr><td>AFNI</td><td>Cox, 1996</td><td>RRID:SCR_005927, http://afni.nimh.nih.gov</td><td></td></tr><tr><td>CO2SMO (ETCO2/SpO2 Monitor), Model 7100</td><td>Novametrix Medical Systems Inc</td><td>N/A</td><td></td></tr><tr><td>Isoflurane</td><td>CP-Pharma</td><td>Cat# 1214</td><td></td></tr><tr><td>Master-9</td><td>A.M.P.I</td><td>N/A</td><td></td></tr><tr><td>Nanoliter Injector</td><td>World Precision Instruments</td><td>Cat# NANOFIL</td><td></td></tr><tr><td>Pancuronium Bromide</td><td>Inresa Arzneimittel</td><td>Cat# 34409.00.00</td><td></td></tr><tr><td>ParaVision 6</td><td>Bruker BioSpin MRI GmbH</td><td>RRID:SCR_001964</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>Cat# 10010-023</td><td></td></tr><tr><td>Rat: Sprague Dawley rat</td><td>Charles River Laboratories</td><td>Crl:CD(SD)</td><td></td></tr><tr><td>SAR-830/AP Ventilator</td><td>CWE</td><td>N/A</td><td></td></tr><tr><td>&amp;alpha;-chloralose</td><td>Sigma-Aldrich</td><td>Cat# C0128-25G;RRID</td><td></td></tr></tbody></table>

real-time fmri brain mapping in animals

动态功能磁共振成像反应在很大程度上取决于动物在麻醉或清醒状态下的生理状况。我们开发了一个实时fMRI平台，指导实验者在采集过程中即时监测fMRI反应，可用于修改动物的生理机能，以实现动物大脑中所需的血流动力学反应。实时功能磁共振成像设置基于14.1T临床前MRI系统，能够实时映射麻醉大鼠初级前爪体感皮层（FP-S1）中的动态fMRI反应。实时fMRI平台不是回顾性分析来研究导致fMRI信号变异性的混杂来源，而是提供了一种更有效的方案，使用定制的宏功能和MRI系统中的通用神经图像分析软件来识别动态fMRI反应。此外，它还为动物的大脑功能研究提供了即时故障排除可行性和实时生物反馈刺激范式。

图 3 和图 4 显示了具有电前爪刺激（3 Hz，4 s，脉冲宽度 300 us，2.5 mA）的代表性实时体素 BOLD-fMRI 时间过程和功能图。fMRI设计范式包括10次刺激前扫描，3次刺激扫描和12次刺激间扫描，总共8个时期（130次扫描）。总扫描时间为 3 分 15 秒（195 秒）。图3显示了实时采集格式中对侧FP-S1的体素时间过程（黑线），对应于块设计范式（红...

Watch this Scientific Journal Video about Real-Time fMRI Brain Mapping in Animals at JoVE.com

Real-Time fMRI Brain Mapping in Animals

动物大脑功能映射可以从实时功能磁共振成像（fMRI）实验设置中受益。利用动物MRI系统中实施的最新软件，我们建立了小动物功能磁共振成像的实时监测平台。

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 ...

Research

JoVE Journal

Neuroscience

3.4K 次观看.  Max Planck Institute for Biological Cybernetics. 动物大脑功能映射可以从实时功能磁共振成像（fMRI）实验设置中受益。利用动物MRI系统中实施的最新软件，我们建立了小动物功能磁共振成像的实时监测平台。

视频: Real-Time fMRI Brain Mapping in Animals

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

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 ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

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 ...

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.

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 ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

大鼠桶内皮层 7 t 的功能磁共振波谱在晶氏体活化过程中的应用

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

免疫与感染

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, neuroanesthesia, and neurosurgery. Patients who have tumors or epileptogenic tissue in eloquent brain areas are studied preoperatively with functional and structural MRI, and intraoperatively with direct electrical stimulation mapping. Post-operative neural and cognitive outcome measures fuel basic science studies about the factors that mediate good versus poor outcome after surgery, and how brain mapping can be further optimized to ensure the best outcome for future patients. In this article, we describe the interdisciplinary workflow that allows our team to meet the synergistic goals of optimizing patient outcome and advancing scientific understanding of the human brain.

This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients.

罗切斯特大学医学中心的转化脑图:通过个性化大脑映射保护大脑

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

使用经颅功能超声成像的小鼠全脑3D激活和功能连接映射

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task state. This protocol describes a preclinical survival method for obtaining rs-fMRI data. Combining low dose isoflurane with continuous infusion of the &#945;2 adrenergic receptor agonist dexmedetomidine provides a robust option for stable, high-quality data acquisition while preserving brain network function. Furthermore, this procedure allows for spontaneous breathing and near-normal physiology in the rat. Additional imaging sequences can be combined with resting-state acquisition creating experimental protocols with anesthetic stability of up to 5 h using this method. This protocol describes the setup of equipment, monitoring of rat physiology during four distinct phases of anesthesia, acquisition of resting-state scans, quality assessment of data, recovery of the animal, and a brief discussion of post-processing data analysis. This protocol can be used across a wide variety of preclinical rodent models to help reveal the resulting brain network changes that occur at rest.

This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

获取大鼠的静态功能磁共振成像数据

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task ...