作者要感谢凯尔吉尔伯特，谁设计的定制射频线圈，和凯文·巴克，是谁设计的MRI兼容雪橇的贡献。这项工作是由健康研究加拿大学院（CIHR），加拿大自然科学和（NSERC）工程研究理事会，加拿大创新基金会（CFI）的支持。

下面的过程可以应用到任何成像实验，其中麻醉猫的使用。这是特别需要的听觉实验（步骤1.1-1.7，2.8，4.1）的步骤可以被修改以适应其它感官刺激协议。 所有实验程序收到来自动物使用小组委员会大学动物保护协会在西安大略大学的批准，随后由加拿大动物保护协会（CCAC）44规定的准则。概述的实验需要约150分钟从动物准备恢复。实验的时间过程示于图1。 1。刺激设备准备 图2示出了电子元件和需要用于产生听觉刺激在MRI扫描仪的相应连接。的要求为follows：一台电脑，一个外置声卡，立体声功放和功能磁共振成像兼容的耳机系统。 <ol><li>连接将用于呈现听觉刺激，通过通用串行总线（USB）电缆的外置声卡的电脑。 </li><li>连接连接外置声卡的输出端口到立体声功率放大器的输入端口的电缆。 </li><li>附加连接立体声功率放大器的输出端口到fMRI的兼容耳机系统的变压器箱的输入端口的电缆。 </li><li>连接双耳式耳机，以在变压器箱的输出端口。 </li><li>使用屏蔽同轴电缆的BNC连接到变压器箱连接到穿透面板扫描仪的房间外面。 </li><li>耳机线缆组件连接到面板渗透率扫描仪房间内的相应的BNC端口。 </li><li>连接泡沫耳塞的耳机，然后连接耳机吨Ø电缆组件。运行测试听觉刺激来确认声音被从计算机发送到耳机。拔下耳机，并在动物准备阶段（步骤2.7）插入泡沫耳塞牢固地插入猫的耳朵。 </li></ol> 2。动物的制备<ol><li>要premedicate猫，通过皮下（SC）注射给药的硫酸阿托品镇静剂混合物（0.02毫克/千克）和乙酰丙嗪（0.02毫克/千克）。 </li><li> 20分钟后，通过肌肉注射（IM）注射诱导麻醉管理氯胺酮（4毫克/千克）和盐酸右美托咪（0.02-0.03毫克/千克）。氯胺酮通常结合了镇静剂和肌肉松弛剂，在这种情况下，右美托咪盐酸盐，以减少震颤和肌强直时氯胺酮单独45使用通常观察到的。这麻醉组合通常导致约150分钟的镇静和常用于兽医实践以诱导麻醉在小动物。 </li><li>一旦猫已经失去了其翻正反射，适用眼药膏的眼睛，以防止在手术过程中干燥。将留置导管的内侧大隐静脉的静脉输送氯胺酮。 </li><li>按捏的前爪然后观察猫是否撤回其爪子脚趾测试成功的麻醉诱导。一旦踏板反射消失，喷洒利多卡因入咽壁，然后插管猫用4.0-4.5气管插管抑制呕吐反射。 </li><li>维持麻醉贯穿了氯胺酮在1-1.5升/分钟输送在100％氧的恒定速率输注（0.6-0.75毫克/千克/小时）和吸入异氟烷（0.4-0.5％）的成像会话。结合在60毫升注射器60毫升盐水和0.07毫升氯胺酮然后将注射器中的注射泵。这个步骤可以premedicating猫之前进行。 </li><li>将温蜡填充加热垫的MRI兼容S的地板导致（ 图3a和3c），那么层周围的雪橇的内壁绝缘塑料泡沫包装。 </li><li>将猫在MRI兼容的雪橇（ 图3c）的绝缘泡沫包装内的胸骨位置。 </li><li>一旦猫被定位，调整头部来访问的耳朵。滚动泡沫耳塞到尽可能最小的直径，然后将每个耳塞深入耳道。一旦插入，泡沫耳塞应该扩大到耳道内填补空间。 </li><li>调整猫直至其头正确地定位在3通道的射频（RF）线圈（ 图3b）内。稳定的脑袋声学阻尼记忆海绵（ 图3d）。将耳朵周围的泡沫提供了扫描仪噪音的附加衰减。 </li><li>包裹猫在绝缘塑料泡沫包装，然后固定和运输的雪橇到扫描仪床上的毯子。 </li><li>输液线，麻醉剂输送管和监控设备连接到猫。将耳机连接到附连到渗透面板的耳机电缆组件。 </li><li>启动氯胺酮输注的0.6毫克/公斤/小时的基流量然后根据需要根据麻醉深度增加流速。设置初始剂量异氟醚0.5％再下降到0.4％，一旦解剖扫描已收集。 </li><li>监视和管理整个用核磁共振成像兼容的监控设备定位在距扫描器孔适当距离的实验记录猫的血氧饱和度，呼气末二氧化碳水平，心脏速率，呼吸和直肠温度（如果可能）。 表1列出了平均值和生理测量成功执行此过程的范围。稳步增加心脏速率和呼吸通常与从麻醉中复苏在即关联。 </li><li>之后的S易讯网络建成后，从雪橇取出的猫。继续提供补充采暖用电热垫和毛巾，直到动物完全恢复。一旦咽反射的回报，取出气管导管。监视猫直到翻正反射恢复则动物返回到设备。在手术后的天评估的动物，以确保从该实验没有不良影响。 </li></ol> 3。脑成像<ol><li>收集猫的大脑解剖扫描的轴向切片方向。使用下面的成像参数解剖参考量：用TR = 750毫秒的FLASH成像序列，TE = 8毫秒，矩阵= 256×256，采集体素大小= 281微米×281微米×1.0毫米。解剖扫描的持续时间是约6分钟。 图4（左面板）提供了一种使用指定的参数获得的样品解剖图像切片。 </li><li>使用该函数的成像参数NAL卷：分段交错平面回波采集（EPI）与TR = 1,000毫秒，TE = 15毫秒，3段/面，21×1毫米的切片;矩阵= 96×96;视野中= 72毫米x 72毫米;采集体素大小为0.75毫米×0.75毫米×1.0毫米，采集时间= 3秒/体积图4（右图）提供使用指定的参数得到的样品功能图像切片。 </li></ol> 4。刺激呈现<ol><li>在其中的听觉刺激播放30秒的块的设计提出了宽带白噪声刺激（0-25千赫，100毫秒连发5毫秒的上升/下降时间，1演示每200毫秒，90-100分贝声压级）和交替用30秒的基线（无刺激）条件（ 图5）。重复此步骤，直到听觉诱发BOLD活动是在听觉皮层观察。各功能运行使用区组设计的持续时间是90卷约4.5分钟。 </li><li>呈现刺激的APpropriate块设计配置功能运行所需的号码。 </li></ol> 5。数据分析<ol><li>选择合适的功能磁共振成像分析软件（ 如 SPM，FSL）来处理收购功能的卷。 </li><li>重新调整各功能音量收购最接近的时间参考解剖扫描量。除使用所产生的动作修正值在步骤5.6。排除任何功能性试验，其中旋转头部运动超过1°或平头动超过1mm。 </li><li> Coregister每个卷的参考解剖扫描。 </li><li>平滑每个卷带2毫米全宽半高（FWHM）高斯滤波器。 </li><li>合并方波（棚车）函数对应于ON-OFF刺激块设计为回归到一般线性模型（GLM）。 </li><li>包括运动校正值不感兴趣的占运动有关的文物回归。申请进行u的p ncorrected统计临界值= 0.001到GLM结果，查看集群的BOLD激活。确定满足修正的最小簇的大小（家庭明智的错误：FWE）P &lt;0.05在集群级别的门槛。设置群集程度阈值这个值来查看统计显著集群地区的利益。 </li><li>在每个体素的过程中在基准块的刺激块和平均BOLD信号的平均BOLD信号之间的差值定义的BOLD信号百分比变化（PSC）。 </li></ol>

作者申报利益，财务或其他方面没有冲突。

在设计的功能磁共振成像实验听觉功能的麻醉动物模型中，以下问题应慎重考虑：麻醉（i）于皮层反应的影响，背景扫描仪噪音（II）的影响，及（iii）优化实验过程的数据采集阶段。 而麻醉制剂生产提供的镇静长时间和功能成像会议期间尽量减少潜在的头部运动的重要优势，麻醉是众所周知的影响皮质的血流动力学。在此协议中描述的麻醉药电（氯胺酮）是常用和猫分别?...

听觉处理哺乳动物目前的了解主要来自侵入性电生理研究在猴子1-5，6-10鼬，蝙蝠11-14，15-19啮齿动物，和猫20-24。电生理技术通常利用细胞外微电极周围的电极尖端的神经组织的一个小的区域之内记录的单个和多个神经元的活动。建立功能成像方法，如光学成像和功能磁共振成像（fMRI）技术，通过提供同步驱动的活动的宏观角度来看整个大脑的多个空间上不同的区域作为有益的补充到细胞外记录。内在信号光学成像有利于通过测量表面组织的反射特性与活动相关的大脑的变化诱发的活动可视化，同时利用功能磁共振成像的血氧水平依赖（BOLD）对比来测量大脑区域的刺激，诱发血流动力学改变一个特定的任务时被激活。光学成像需要的皮质表面的直接接触措施表面组织反射率有关系的刺激诱 ​​发的活动25的变化。相比较而言，功能磁共振成像是无创和利用的缺氧血顺磁特性，以在一个完整的颅骨测量两个皮质表面26-28和沟系27,29诱发活性。在非人灵长类视觉皮层30和人类听觉皮层31的BOLD信号和神经活动之间的强相关性验证的功能磁共振成像研究感觉功能的有用工具。由于磁共振成像已被广泛用于研究听觉通路如tonotopic组织32-36，听觉功能37的不对称，皮层激活的模式，识别皮层区域38，声音效果的特征在听反应特性的39,40，和大胆的响应时间当然29,41在人，猴和大鼠模型，合适的功能成像协议，研究听觉功能在猫的发展将提供一个有益的补充的强度特性该功能成像文学。而功能磁共振成像也被用来探索在被麻醉的猫26-28,42视觉皮层的各种功能方面，一些研究已经使用这种技术来检查感觉处理在猫听觉皮层。本议定书的目的是建立利用fMRI来量化函数在麻醉猫的听觉皮层的有效方法。在这个手稿中概述的程序已经实验成功地用来形容在成年猫的听觉皮层43的BOLD响应时间过程的功能。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>Atropine sulphate injection 0.5 mg/mL</td>
 <td>Rafter 8 Products</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Acepromazine 5 mg/mL</td>
 <td>Vetoquinol Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine hydrochloride 100 mg/mL</td>
 <td>Bimeda-MTC</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dexmedetomidine hydrochloride (Dexdomitor 0.5 mg/mL)</td>
 <td>Orion Pharma</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane 99.9%</td>
 <td>Abbott Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lidocaine (Xylocaine endotracheal 10 mg/metered dose)</td>
 <td>Astra Zeneca</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lubricating opthalmic ointment (Refresh Lacri Lube)</td>
 <td>Allergan Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Saline 0.95%</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>IV Catheter 22g (wings)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>IV Extension Set</td>
 <td>Codan US Corp.</td>
 <td>BC 269</td>
 <td></td>
 </tr>
 <tr>
 <td>IV Administration Set 10 drips/mL</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Endotracheal tube 4.0</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pads (Snuggle Safe)</td>
 <td>Lenric C21 Ltd.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe 60 mL</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>External sound card</td>
 <td>Roland Corporation</td>
 <td>Cakewalk UA-25EX</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereo power amplifier</td>
 <td>Pyle Audio Inc.</td>
 <td>Pyle Pro PCAU11</td>
 <td></td>
 </tr>
 <tr>
 <td>MRI-compatible insert earphone system</td>
 <td>Sensimetric Corporation</td>
 <td>Model S14</td>
 <td></td>
 </tr>
 <tr>
 <td>Foam ear tips for insert earphones</td>
 <td>E-A-R Auditory Systems</td>
 <td>Earlink 3B</td>
 <td></td>
 </tr>
 <tr>
 <td>End-tidal CO2 monitor</td>
 <td>Nellcor&nbsp;</td>
 <td>N-85</td>
 <td></td>
 </tr>
 <tr>
 <td>MRI-compatible pulse oximeter</td>
 <td>Nonin Medical Inc.</td>
 <td>Model 7500</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe pump</td>
 <td>Harvard Apparatus</td>
 <td>70-2208</td>
 <td></td>
 </tr>
 </tbody>
 </table>

functional imaging of auditory cortex in adult cats using high-field fmri

感觉处理的哺乳动物听觉系统现有的知识主要来自于各种动物模型的电生理研究，包括猴子，白鼬，蝙蝠，老鼠和猫。为了画听觉功能的人类和动物模型之间合适的相似之处，它可以建立人体功能成像研究和动物的电生理研究之间的桥梁是非常重要的。功能磁共振成像（fMRI）是测量整个大脑皮层的不同区域广泛血流动力学活动模式的建立，微创的方法。这种技术被广泛用于探测在人类大脑感觉功能，是在连接听觉处理的研究在人类和动物的有用工具，并已成功地用于研究在猴和啮齿动物的听觉功能。以下协议描述了一个实验过程中麻醉的成人调查听觉功能猫用功能磁共振成像测量听觉皮层刺激诱发血流动力学改变。这种方法有利于在不同的车型听觉功能的血流动力学反应从而导致更好地理解哺乳动物的听觉皮层的种类无关的特性比较。

代表功能的数据是在一个7T水平孔扫描仪获取并使用统计参数映射工具箱在MATLAB中进行分析。在猫中使用所描述的实验方案43健壮皮质的血液动力学反应，听觉刺激一直被观察到。 图6示出了BOLD激活在2只动物在响应于一个30秒的宽带噪声刺激在一个块中的设计呈现。宽带噪声与基线（无刺激）的T型统计图2中的图像切片平面的对比揭示了在听觉皮层（ 图6a和6d...

Watch this Scientific Journal Video about Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI at JoVE.com

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

听觉系统在哺乳动物中的功能研究传统上使用诸如电生理记录空间为重点的技术进行的。以下协议描述可视化诱发血流动力学行为的大型图案的猫听觉皮层使用功能性磁共振成像的方法。

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 ...

functional-imaging-auditory-cortex-adult-cats-using-high-field

Research

JoVE Journal

Neuroscience

11.4K 次观看.  University of Western Ontario. 听觉系统在哺乳动物中的功能研究传统上使用诸如电生理记录空间为重点的技术进行的。以下协议描述可视化诱发血流动力学行为的大型图案的猫听觉皮层使用功能性磁共振成像的方法。 

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

关联从人类前额叶皮层的fMRI信号的行为反应：检查的认知过程，使用任务分析

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

使用功能磁共振成像和弥散加权成像脑结构与功能分析

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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

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

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

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

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

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

Real-Time fMRI Brain Mapping in Animals

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

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

动物实时功能磁共振成像大脑映射

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

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

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

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

使用经颅功能超声成像的小鼠全脑3D激活和功能连接映射

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