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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+respiratory-variation-related+neuronal-activity-related+fluctuations+in+fluctuations+from+fMRI.">Separating respiratory-variation-related neuronal-activity-related fluctuations in fluctuations from fMRI.</a> Neuroimage. 31 (4), 1536-1548 (2006).</li><li>Golestani, A. M., Chang, C., Kwinta, J. B., Khatamian, Y. B., Chen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+end-tidal+CO2+response+function+in+the+resting-state+BOLD+fMRI+signal:+Spatial+specificity,+test-retest+reliability+and+effect+of+fMRI+sampling+rate.">Mapping the end-tidal CO2 response function in the resting-state BOLD fMRI signal: Spatial specificity, test-retest reliability and effect of fMRI sampling rate.</a> Neuroimage. 104, 266-277 (2015).</li><li>Lu, H. 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. J. <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+reconstruction+and+high-speed+processing+in+functional+MR+imaging.">Real-time reconstruction and high-speed processing in functional MR imaging.</a> American Journal of Neuroradiology. 19 (7), 1297-1300 (1998).</li><li>Voyvodic, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+fMRI+paradigm+control,+physiology,+and+behavior+combined+with+near+real-time+statistical+analysis.">Real-time fMRI paradigm control, physiology, and behavior combined with near real-time statistical analysis.</a> Neuroimage. 10 (2), 91-106 (1999).</li><li>Cohen, M. 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. 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=Ultra+high-resolution+fMRI+and+electrophysiology+of+the+rat+primary+somatosensory+cortex.">Ultra high-resolution fMRI and electrophysiology of the rat primary somatosensory cortex.</a> Neuroimage. 73, 113-120 (2013).</li><li>Masamoto, K., Kim, T., Fukuda, M., Wang, P., Kim, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+neural,+vascular,+and+BOLD+signals+in+isoflurane-anesthetized+rat+somatosensory+cortex.">Relationship between neural, vascular, and BOLD signals in isoflurane-anesthetized rat somatosensory cortex.</a> Cerebral Cortex. 17 (4), 942-950 (2007).</li><li>van Alst, T. 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. H., Zhang, Y., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+change+of+functional+connectivity+specificity+in+rats+under+various+anesthesia+levels+and+its+neural+origin.">The change of functional connectivity specificity in rats under various anesthesia levels and its neural origin.</a> Brain Topography. 26 (3), 363-377 (2013).</li><li>Liu, X. P., 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=Multiphasic+modification+of+intrinsic+functional+connectivity+of+the+rat+brain+during+increasing+levels+of+propofol.">Multiphasic modification of intrinsic functional connectivity of the rat brain during increasing levels of propofol.</a> Neuroimage. 83, 581-592 (2013).</li><li>Hutchison, R. M., Hutchison, M., Manning, K. Y., Menon, R. S., Everling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+dose-dependent+alterations+in+the+cortical+connectivity+profiles+and+dynamic+properties+of+the+brain's+functional+architecture.">Isoflurane induces dose-dependent alterations in the cortical connectivity profiles and dynamic properties of the brain's functional architecture.</a> Human Brain Mapping. 35 (12), 5754-5775 (2014).</li><li>He, 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=Ultra-Slow+Single-Vessel+BOLD+and+CBV-Based+fMRI+Spatiotemporal+Dynamics+and+Their+Correlation+with+Neuronal+Intracellular+Calcium+Signals.">Ultra-Slow Single-Vessel BOLD and CBV-Based fMRI Spatiotemporal Dynamics and Their Correlation with Neuronal Intracellular Calcium Signals.</a> Neuron. 97 (4), 925-939 (2018).</li><li>Yoshida, K., 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=Physiological+effects+of+a+habituation+procedure+for+functional+MRI+in+awake+mice+using+a+cryogenic+radiofrequency+probe.">Physiological effects of a habituation procedure for functional MRI in awake mice using a cryogenic radiofrequency probe.</a> Journal of Neuroscience Methods. 274, 38-48 (2016).</li></ol>

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>&#945;-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

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: 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.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

神经科学的研究和实时功能皮层映射记录人力Electrocorticographic（ECOG）信号

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

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 ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...