这项工作部分得到了日本科学促进会（JSPS）科研资助（C）18K08956和福冈大学中央研究所（第201045号）的资助。

这项研究得到了日本福冈大学机构审查委员会（IRB）的批准（IRB No. 2017M017）。在参与之前，所有患者都提供书面知情同意。
1. 准备 fNIRS 实验
注：本实验使用了多通道连续波激光基NIRS系统。近红外光的波长为780纳米、805纳米和830纳米，采样率为7.8赫兹。时间和空间分辨率（发射光器和探测器探测器之间的距离）分别为0.13 s和3.0厘米。
<ol><li>将 fNIRS 设备设置在一个黑暗无噪音的地方。在室温下进行实验。在实验前30分钟启动fNIRS仪器。 注：fNIRS系统在受控温度和湿度条件下使用，以确保设备稳定运行。</li>
	<li>使用整个头盖进行 fNIRS 记录，并将头盖固定在主体的头上，以便对应于国际 10-20 系统中央 （Cz） 的位置位于头盖第 245 号的支架上。（图1） 。</li>
	<li>将标记贴纸附在参考位置点：鼻腔 （Nz）、右外部听觉肉 （AR） 和左外部听觉肉 （AL）。 注：由于三维 （3D） 坐标围绕 Nz、AR、AL 标记贴纸和 Cz 支架的位置进行读取，因此在拍照前必须附加标记贴纸。</li>
	<li>在高分辨率数码相机进行空间注册校准后，用探头位置拍摄受试者头部的照片，同时从 15 个角度显示参考点（Cz、Nz、AR 和 LR）。 注意：请在放置探头之前拍照。如果放置探头后拍摄照片，则探头和布线可能隐藏标记地标。根据制造商的建议，在拍摄 12 张照片 30° 对角线向前到主体的右侧后，在稍微高于 3 张或更多照片，使 Cz（持有人 No. 245）出现在图片中。这是因为在总共拍摄 15 张或更多张照片时，很容易使其成为三维拍摄。</li>
	<li>使用发光二极管 （LED） 照明塑料棒连接探头，小心地分离受试者干扰光极的头发。安排探头，使光极连接到从头皮表面的最小距离，并与头皮接触。 注意：仔细检查是否有任何压力或不适的病人由于附件的光子，因为增加强度的全身混淆与自主神经系统激活23。</li>
	<li>将带有 32 个光极（16 个光源和 16 个探测器;每个半球 4 x 4 个阵列）的 48 通道系统安排在正面和相干区域作为感兴趣区域的头部盖上（图 2）。</li>
	<li>开始并使用 3D 数字化软件来确定空间注册。</li>
	<li>扫描整个头部的图片数据后，通过自动测量确定每个患者的空间坐标，并保存为 "起源 " 文件和其他 文件（*CSV 文件）。 注意：如果无法使用自动测量从图像中检测到坐标点，请手动输入调整。</li>
</ol>2. 运行实验
<ol><li>为实验选择块设计，任务可以是研究的任何兴趣运动，如手开/闭，手指敲击等。在我们以前的研究中，任务是机器人辅助肘部运动15。 注：每个周期由三个方块（15 s 休息 - 15 s 任务 - 15 s 休息）组成，每个患者在每个会话中完成 7 个周期。</li>
	<li>让参与者在舒适的位置等待，直到启动信号。指示受试者在休息和任务期间闭上眼睛。</li>
	<li>给开始和停止提示（即"重复弯曲和肘部延伸"，"停止和放松"）。 注意：测量时不要说话。测量期间，仔细检查监视器屏幕上是否有人工制品。</li>
	<li>以相同的姿势执行块设计任务。直立姿势与站立或坐姿是可取的，不要扭曲耳机。 注意：如果患者在长时间佩戴头架后感到不舒服，请在机器人辅助练习中取出或松开探头。</li>
	<li>完成 NIRS 测量后，取下头架并标记贴纸以结束实验。 注意：仔细检查由于长时间佩戴对头皮的皮肤损伤。</li>
</ol>3. 使用 NIRS-SPM 软件进行定性 GLM 分析
<ol><li>在马特拉布软件上启动 NIRS-SPM。将与从 NIRS 设备获得的 HbO2 和 HHb 浓度变化相关的数据文件转换为用于 NIRS-SPM 分析的文件格式。</li>
	<li>从弹出式菜单中选择使用 NIRS 系统的选项。选择加载按钮并选择转换的 HbO2 和 HHb 浓度更改选项。</li>
	<li>检测 NIRS 通道位置的空间注册。选择 独立 复选框，然后选择 带 3D 数字化复 选框。</li>
	<li>在从真实坐标到 MNI 空间中，使用对话框选择 _origin。CSV 是指坐标参考点文件， 并_others。CSV 是指坐标探头/通道文件。</li>
	<li>选择 "注册 "按钮。选择要进行空间估计的点，然后单击 "确定 "按钮。单击 "渲染大脑"按钮的项目 MNI 坐标 。 注：根据蒙特利尔神经研究所 （MNI） 大脑模板估计 NIRS 通道位置的空间位置。</li>
	<li>选择 "多萨尔视图" 选项，然后单击 "保存" 按钮。</li>
	<li>在指定第 1级部分时，请选择 NIRS 数据文件名和 SPM 目录。选择血红蛋白复选框;HbO2或HHb.突出显示指定设计选项并选择Sec选项。突出显示条件/试验选项的数量，并输入数字7。</li>
	<li>突出显示发病和持续时间选项的矢量，并输入按以下实验条件持续时间乘以的发病媒介。 注意：在这种情况下，发病时间的向量应指定为[15：45：285] 或 [15 60 105 150 195 240 285]。持续时间的向量应指定为[15* （7，1）] 或 [15 15 15 15 15 15 15] 。</li>
	<li>要进行去趋势化，请选择 波莱特-MDL 按钮。使用 预着色方法：低通滤清器 并选择 hrf 按钮，并纠正串行相关性，然后选择 无 按钮。</li>
	<li>在估计时间相关性时，请检查 个人分析。 注意：保存单独的 MNI 坐标系统中 fNIRS 通道位置的空间本地化作为文本文件。同样，将地图保存在基于单个布罗德曼区域 （BA） 作为文本文件。</li>
	<li>在估计时间相关性时，请查看 组分析。NIRS-SPM 根据 MNI 标准化的大脑坐标系统对参与者人数的平均光学位置进行对齐。</li>
	<li>根据标准化大脑血红蛋白水平的变化计算激活图。HbO2 和 HHb 水平在 p < 0.01 的未更正阈值下被认为显著。 注：左/右信息在受右影响的侧翻转，用于组分析。</li>
</ol>4. 基于分层混合模型的多渠道比较分析
<ol><li>启动 SAS 软件。转换文本文档（。使用低通路滤波器处理的 NIRS 数据文件中 HbO2 和 HHb 的浓度变化的 TXT（截止频率设置为 0.1 Hz）到电子表格软件逗号分离值文件（）。CSV）。</li>
	<li>使用程序创建导入 SAS 数据 （.sas7bdat）。</li>
	<li>输出导入文件，并下列命令，以"导入文件"为名</li>
	<li>为每个主题输出干预前文件，在分析 SAS 中运行以下命令。（图3）在创建导入文件时，给出一个可以识别主题信息和干预前与干预后（例如 id1 预，id1 帖子）的名称。。。</li>
	<li>运行每个通道的干预前和干预后数据命令（ch1-48;HbO2 和 HHb 如下所示 （图 4）。</li>
	<li>根据从输出结果中获得的数据，在电子表格工作簿文件（.xlsx）中输入每个通道的干预前差异（任务和休息差异）、休息和任务值（估计值、上限和下限）。</li>
	<li>同样，将固定效果类型 3 测试的交互项的自由度、F 值和 P 值的数字和分母度输入到电子表格工作簿文件（.xlsx）中。</li>
	<li>要控制多通道测试中的误发现率 （FDR），请使用本杰明和霍奇伯格方法27， 并将 FDR 控制在 p 值< 0.01。</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+functional+near-infrared+spectroscopy+(fNIRS)+neuroimaging+in+exercise(-)cognition+science:+A+systematic,+methodology-focused+review.">Applications of functional near-infrared spectroscopy (fNIRS) neuroimaging in exercise(-)cognition science: A systematic, methodology-focused review.</a> Journal of Clinial Medicine. 7 (12), (2018).</li><li>Boden, 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=The+oxygenation+response+to+functional+stimulation:+is+there+a+physiological+meaning+to+the+lag+between+parameters.">The oxygenation response to functional stimulation: is there a physiological meaning to the lag between parameters.</a> Neuroimage. 36 (1), 100-107 (2007).</li><li>Pinti, P., Scholkmann, F., Hamilton, A., Burgess, P., Tachtsidis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+and+issues+regarding+pre-processing+of+fNIRS+neuroimaging+data:+An+investigation+of+diverse+signal+filtering+Methods+within+a+general+linear+model+Framework.">Current status and issues regarding pre-processing of fNIRS neuroimaging data: An investigation of diverse signal filtering Methods within a general linear model Framework.</a> Frontiers in Human Neuroscience. 12, 505 (2018).</li><li>Udina, C., 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=Functional+near-infrared+spectroscopy+to+study+cerebral+hemodynamics+in+older+adults+during+cognitive+and+motor+tasks:+a+review.">Functional near-infrared spectroscopy to study cerebral hemodynamics in older adults during cognitive and motor tasks: a review.</a> Frontiers in Aging Neuroscience. 11, 367 (2019).</li><li>Verghese, J., Wang, C., Ayers, E., Izzetoglu, M., Holtzer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+activation+in+high-functioning+older+adults+and+falls:+Prospective+cohort+study.">Brain activation in high-functioning older adults and falls: Prospective cohort study.</a> Neurology. 88 (2), 191-197 (2017).</li><li>Yucel, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+separation+regression+improves+statistical+significance+and+better+localizes+the+hemodynamic+response+obtained+by+near-infrared+spectroscopy+for+tasks+with+differing+autonomic+responses.">Short separation regression improves statistical significance and better localizes the hemodynamic response obtained by near-infrared spectroscopy for tasks with differing autonomic responses.</a> Neurophotonics. 2 (3), 035005 (2015).</li><li>Torricelli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+domain+functional+NIRS+imaging+for+human+brain+mapping.">Time domain functional NIRS imaging for human brain mapping.</a> Neuroimage. 85, 28-50 (2014).</li><li>Giacalone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-domain+near-infrared+spectroscopy+in+acute+ischemic+stroke+patients.">Time-domain near-infrared spectroscopy in acute ischemic stroke patients.</a> Neurophotonics. 6 (1), 015003 (2019).</li><li>Saita, 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=Contralateral+cerebral+hypometabolism+after+cerebellar+stroke:+a+functional+near-infrared+spectroscopy+study.">Contralateral cerebral hypometabolism after cerebellar stroke: a functional near-infrared spectroscopy study.</a> Journal of Stroke and Cerebrovascular Diseases. 26 (4), 69-71 (2017).</li></ol>

作者与本研究无关，不得披露利益冲突。

在我们的 fNIRS 小组分析方法中，除了通过定性 t统计映射执行成像分析方法外，我们还使用比较多通道分析比较干预前与干预后（机器人辅助练习）。在定性分析方面，我们使用 NIRS-SPM 软件作为基于通用线性模型的大规模不变方法。NIRS-SPM 分析通过在任务期间可视化激活区域来显示每个会话的定性结果。此外，非侵入性 3D 数字化器的信息能够估计 fNIRS 通道相对于大脑的位置。使用NIRS-SPM?...

神经康复在感官运动障碍后的功能恢复中起着重要作用。为了澄清神经可塑性相关功能恢复的机制，我们使用了各种神经成像技术，如功能磁共振成像 （fMRI）、负电子发射断层扫描 （PET）、脑电图 （EEG） 和功能近红外光谱 （fNIRS）。不同的成像模式有不同的优点和缺点。虽然fMRI是最典型的设备，它受磁场的影响，具有高成本，高物理限制，和有限的感官运动任务1，2，3，4。fNIRS 设备是一种非侵入性光学神经成像，空间分辨率相对较低，但与 fMRI4相比，它具有更好的时间分辨率。fNIRS在验证治疗效果时是合适的，因为它比较了干预前和干预后的效果，具有动态运动任务，便携，在自然环境中比fMRI1、2、4功能更显著。据报道，NIRS在脑血管疾病、癫痫病、严重脑损伤、帕金森病和认知障碍等领域更为适用。在感官运动任务方面，它广泛应用于步态和站立平衡6、7、8、上肢功能（抓手、手指敲击）8、9、复杂运动技能训练10、11、机器人12、13、14、15和脑-计算机界面16、17、18。fNIRS基于光学神经成像和神经血管耦合原理，测量皮质代谢活动，增加血液流动，从而皮质活性作为次要信号19。据报道，fNIRS信号与血氧水平依赖性fMRI20信号有很强的相关性。连续波 fNIRS 使用修改后的啤酒-兰伯特定律，根据宽带近红外光衰减21、22的测量变化，确定含氧血红蛋白 （HbO2）和脱氧血红蛋白 （HHb） 皮质浓度水平的变化。由于无法使用连续波 NIRS 系统测量微分路径长度因子 （DPF），因此我们假设 DPF 是恒定的，血红蛋白信号变化表示为任意单位毫米 （mM x mm）2，18。
fNIRS 实验需要选择最充分的方法，包括探针设置、实验设计和分析方法。在探针设置方面，EEG测量中采用的国际10-20方法是许多研究人员在神经成像中使用的设定标准。近年来，根据蒙特利尔神经学研究所（MNI）坐标，基于标准大脑的坐标设置被使用。该实验使用块设计，通常用于感官运动任务，以及事件相关设计。这是比较休息和任务期间血红蛋白浓度变化的方法：HbO2 浓度水平增加，HHb 浓度水平随着与任务依赖皮质活动相关的脑血流量变化而降低。虽然有各种分析方法，但 NIRS-SPM 免费软件能够进行类似于 fMRI 统计参数映射 （SPM） 的分析。NIRS 数据的处理使用基于一般线性模型 （GLM） 的大规模不变方法。在执行任务依赖性大脑活动分析时，fNIRS 测量可受脑部和脑外隔间23中唤起或非唤起的神经元活动和全身生理干扰（心率、血压、呼吸速率和自主神经系统活动）的影响。因此，预分析处理、过滤、波转换和主要组件分析是有用的。关于使用 NIRS-SPM 进行数据处理的过滤和人工制品，使用低通滤9 和波段最小描述长度 （Wavelet-MDL）24 去趋势来克服运动或其他噪声/神器来源。有关此分析方法的详细信息，请参阅 Ye 等人25 的报告。虽然只有使用 SPM 的报告，但它只是图像分析的定性指标，由于 NIRS 的空间分辨率较低，因此组分析需要极其谨慎。此外，当 DPF 是恒定的时，不应执行通道和个人之间的数字比较，但可以验证每个通道的变化差异。基于上述条件，为了补充NIRS-SPM组分析结果，在提高空间注册精度后，采用了原有的多通道分析分析方法。这种多渠道分析比较了治疗前后每个通道的 HbO2 和 HHb 水平变化幅度，使用具有固定干预（之前或之后）、固定周期（休息或任务）和随机单个效果的分层混合模型。
这样，有几个fNIRS测量和分析方法：但是，尚未确定标准方法。本文介绍了基于GLM的定性统计参数映射和比较多层次的分层混合模型，利用具有感官运动任务的块设计，分析多通道fNIRS干预前与干预后实验获得的数据。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D-digitizer software</td>
			<td>TOPCON</td>
			<td>-</td>
			<td>NS-1000 software ver.1.50</td>
		</tr>
		<tr>
			<td>NIRS system</td>
			<td>Shimadzu</td>
			<td>-</td>
			<td>FOIRE-3000</td>
		</tr>
		<tr>
			<td>Robot</td>
			<td>CYBERDYNE</td>
			<td>-</td>
			<td>Single-joint type Hybrid Assitive Limb (HAL-SJ)</td>
		</tr>
	</tbody>
</table>

qualitative and comparative cortical activity data analyses from a functional near-infrared spectroscopy experiment applying block design

神经成像研究在评估介入前神经系统疾病（如康复和手术治疗）方面起着关键作用。在用于测量大脑活动的众多神经成像技术中，功能近红外光谱 （fNIRS） 通过测量类似于功能磁共振成像 （fMRI） 的局部血红蛋白水平，能够评估动态皮质活动。此外，由于 fNIRS 中的物理限制较小，可以评估感官运动任务的多个变体。许多实验室已经开发出几种用于fNIRS数据分析的方法：然而，尽管一般原则是相同的，但没有普遍标准化的方法。在这里，我们介绍了使用块设计从多通道 fNIRS 实验中获得的数据的定性和比较分析方法。在定性分析方面，我们使用 NIRS 软件作为基于通用线性模型的大规模不变方法。NIRS-SPM 分析通过在任务期间可视化激活区域来显示每个会话的定性结果。此外，非侵入性三维数字化器可用于估计 fNIRS 通道相对于大脑的位置。为了证实NIRS-SPM的发现，可以通过比较使用多通道分层混合模型从同一研究对象的两个不同会话（干预前后）获得的数据进行统计分析。我们的方法可用于测量各种神经系统疾病（如运动障碍、脑血管疾病和神经精神病）的干预前与干预后分析。

在此，我们介绍了我们团队目前正在研究的机器人辅助康复：急性中风患者上肢运动缺陷的生物反馈效应。我们包括10名同意中风患者（平均年龄：66.8岁±12.0岁;两名妇女和8名男子）住进我们医院。在下肢中风阶段，在发病后2周多，我们使用fNIRS系统评估了这些患者在同一天上肢机器人辅助康复前后与运动相关的皮质活动。关于块设计任务，他们在每个任务周期中在 15s 内...

Watch this Scientific Journal Video about Qualitative and Comparative Cortical Activity Data Analyses from a Functional Near-Infrared Spectroscopy Experiment Applying Block Design at JoVE.com

Qualitative and Comparative Cortical Activity Data Analyses from a Functional Near-Infrared Spectroscopy Experiment Applying Block Design

我们使用具有感官运动任务的块设计描述连续波功能近红外光谱实验的分析。为提高数据分析的可靠性，我们采用了基于定性的一般线性模型统计参数映射和多通道比较分层混合模型。

功能近红外光谱实验应用块设计的定性和比较皮质活动数据分析

Neuroimaging studies play a pivotal role in the evaluation of pre- vs. post-interventional neurological conditions such as in rehabilitation and surgical ...

qualitative-comparative-cortical-activity-data-analyses-from

Research

JoVE Journal

Neuroscience

3.6K 次观看.  Fukuoka University. 我们使用具有感官运动任务的块设计描述连续波功能近红外光谱实验的分析。为提高数据分析的可靠性，我们采用了基于定性的一般线性模型统计参数映射和多通道比较分层混合模型。

视频: Qualitative and Comparative Cortical Activity Data Analyses from a Functional Near-Infrared Spectroscopy Experiment Applying Block Design

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

使用近红外光谱MazeSuite和功能研究学习空间导航

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

科研

神经科学

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

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), ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in remote locations. This paper shows the development of field functional Near Infrared Spectroscopy (fNIRS) imaging to the study of child language, reading, and cognitive development in a rural village setting of C&#244;te d&#39;Ivoire. Innovation in methods and the development of culturally appropriate neuroimaging protocols allow&#160;a first-time look into the brain&#39;s development and children&#39;s learning outcomes in understudied environments. This paper demonstrates protocols for transporting and setting up a mobile laboratory, discusses considerations for field versus laboratory neuroimaging, and presents a guide for developing neuroimaging consent procedures and building meaningful long-term collaborations with local government and science partners. Portable neuroimaging methods can be used to study complex child development contexts, including the impact of significant poverty and adversity on brain development. The protocol presented here has been developed for use in C&#244;te d&#39;Ivoire, the world&#39;s primary source of cocoa, and where reports of child labor in the cocoa sector are common. Yet, little is known about the impact of child labor on brain development and learning. Field neuroimaging methods have the potential to yield new insights into such urgent issues, and the development of children globally.

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy NIRS Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural C&#244;te d&#39;Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain&#39;s development and children&#39;s learning outcomes in environments with significant poverty and adversity.

利用功能近红外光谱成像技术研究全球儿童发展: 农村撒哈拉以南非洲地区的神经影像场方法

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in ...

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding of the neurobiological underpinnings of social interactions, and possibly interpersonal relationships. Functional near-infrared spectroscopy (fNIRS) is well suited for conducting hyperscanning experiments because it measures local hemodynamic effects with a high sampling rate and, importantly, it can be applied in natural settings, not requiring strict motion restrictions. In this article, we present a protocol for conducting fNIRS hyperscanning experiments with parent-child dyads and for analyzing brain-to-brain synchrony. Furthermore, we discuss critical issues and future directions, regarding the experimental design, spatial registration of the fNIRS channels, physiological influences and data analysis methods. The described protocol is not specific to parent-child dyads, but can be applied to a variety of different dyadic constellations, such as adult strangers, romantic partners or siblings. To conclude, fNIRS hyperscanning has the potential to yield new insights into the dynamics of the ongoing social interaction, which possibly go beyond what can be studied by examining the activities of individual brains.

The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions.

利用功能近红外光谱进行超扫描实验

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding ...

Conducting Concurrent Electroencephalography and Functional Near-Infrared Spectroscopy Recordings with a Flanker Task

Concurrent EEG and fNIRS recordings offer an excellent opportunity to gain a full understanding of the neural mechanism of cognitive processing by inspecting the relationship between the neural and hemodynamic signals. EEG is an electrophysiological technology that can measure the rapid neuronal activity of the cortex, whereas fNIRS relies on the hemodynamic responses to infer brain activation. The combination of EEG and fNIRS neuroimaging techniques can identify more features and reveal more information associated with the functioning of the brain. In this protocol, fused EEG-fNIRS measurements were performed for concurrent recordings of evoked-electrical potentials and hemodynamic responses during a Flanker task. In addition, the critical steps for setting up the hardware and software system as well as the procedures for data acquisition and analysis were provided and discussed in detail. It is expected that the present protocol can pave a new avenue for improving the understanding of the neural mechanisms underlying various cognitive processes by using the EEG and fNIRS signals.

The present protocol describes how to perform concurrent EEG and fNIRS recordings and how to inspect the relationship between the EEG and fNIRS data.&#160;

使用 Flanker 任务进行并发脑电图和功能近红外光谱记录

Concurrent EEG and fNIRS recordings offer an excellent opportunity to gain a full understanding of the neural mechanism of cognitive processing by ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

肌动蛋白动力学、离合器耦合和牵引力分析，促进生长锥体前进

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

Group Synchronization During Collaborative Drawing Using Functional Near-Infrared Spectroscopy

Functional near-infrared spectroscopy (fNIRS) is a noninvasive method particularly suitable for measuring cerebral cortex activation in multiple subjects, which is relevant for studying group interpersonal interactions in ecological settings. Although many fNIRS systems technically offer the possibility to monitor more than two individuals simultaneously, establishing easy-to-implement setup procedures and reliable paradigms to track hemodynamic and behavioral responses in group interaction is still required. The present protocol combines fNIRS and video-based observation to measure interpersonal synchronization in quartets during a cooperative task.This protocol provides practical recommendations for data acquisition and paradigm design, as well as guiding principles for an illustrative data analysis example. The procedure is designed to assess differences in brain and behavior interpersonal responses between social and non-social conditions inspired by a well-known ice-breaker activity, the Collaborative Face Drawing Task. The described procedures can guide future studies to adapt group naturalistic social interaction activities to the fNIRS environment.

The present protocol combines functional near-infrared spectroscopy (fNIRS) and video-based observationto measure interpersonal synchronization in quartets during a collaborative drawing task.

使用功能近红外光谱的协作绘图期间的组同步

Functional near-infrared spectroscopy (fNIRS) is a noninvasive method particularly suitable for measuring cerebral cortex activation in multiple subjects, ...

Methods Article

功能近红外光谱实验应用块设计的定性和比较皮质活动数据分析