这项工作是由美国国立卫生研究院支持1 R21NS057718 - 01。

1。这是一个非生存手术。第一步是植入电极。在这个例子中，将植入电极在两个半球的初级躯体感觉皮层的前爪地区。 <ol><li> 2％异氟醚麻醉大鼠（雄性SD大鼠，200-300克），立体定向手术系统修复到位。手术开始之前，确保动物麻醉，并没有一个脚趾头掐的响应展品。打开头皮之前删除的皮毛。上述颅骨的肌肉和其他组织分离，并阻止任何出血，骨表面使用cauterizer。 </li><li>码头准备在头骨表面（靠近中线的前V形路口）作为植入电极轴固定点，用牙科水泥。设置一个小尼龙螺丝设置成骨前用牙科水泥可能会增加稳定性。的形状与水泥码头大小应在面积约5毫米高，3 × 5毫米2的基地（见图1） 。 </li><li>用细尖的电气钻，小心地打开颅骨，并在每个半球的初级躯体感觉皮层的前爪代表性，暴露硬脑膜。每个洞的直径约1毫米，定位1毫米前，从囟门外侧为4毫米。在显微镜下，切开一个微小的开放，用注射器针头在硬脑膜，小心地避免任​​何船只受损。 </li><li>在每个电极插入，确保附近的切口，无出血或渗出物。玻璃微电极应做好术前约3〜4厘米轴的长度和阻抗的1〜5MΩ。与人工脑脊液（学联）填充电极的毛细管插入每个电极斜刺入脑〜0.4毫米，采用立体定向手臂打开硬脑膜（〜45 °，从后到前）。固定到位之前，检查的电信号。一个氯化银线的一端浸入到学联，另一端连接到输入放大器。一个银线，连接在后面打开皮肤皮下，作为参比电极。 </li><li>电极固定前，仔细检查手术区，并确保没有发生出血或渗出，然后适用于牙膏，以取代删除的皮肤和肌肉的头骨。牙膏的使用提高了MR图像质量，减少在颅骨/空气界面的易感性不匹配。将电极轴，以准备与牙科水泥码头（见图1）。 </li><li>牙科水泥固化后，动物转移的MRI的摇篮和修复到位。监测大鼠为研究的其余部分，包括体温，呼吸率，SPO 2和心率的生理条件。 </li><li>位置在头部表面线圈（发送/接收），与电极线圈的中心凸出。作为支持固定电极导线，以避免引起动物呼吸运动的另外一个拱状的硬盖位于上盖的摇篮。导致用于同时成像和记录扩展到〜5米（放大器是位于外面的磁铁室），并与导电塑料作为一个被动的盾覆盖。 </li><li> medetomidine减少抑制神经活动，如果需要，可以切换从异氟醚麻醉。一个动物转移到磁铁之前的最后一次检查的电信号。在我们的研究中，记录参数如下：× 1000放大，为0.1Hz〜5 K赫兹带通滤波，60赫兹陷波过滤，12 kHz的采样率模拟到数字转换。 </li></ol> 2。在这一点上，动物是插入核磁共振成像扫描仪的同时成像和记录。动物是整个成像过程中麻醉。 <ol><li> 9.4 T小动物磁共振成像系统（布鲁克，德国）是用在我们的研究。录音之前，成像参数必须建立。一个三个平面侦察员形象是用来定位的功能磁共振成像扫描。为了提高磁场的均匀性，利益的体积匀使用FASTMAP 1。 fMRI研究，日冕成像片入选，其中包括双边前爪初级躯体感觉区，在其中植入电极。外延成像参数视场，1.92 × 1.92厘米2;矩阵的大小，64 × 64;平面分辨率，0.3 × 0.3毫米2;层厚，2毫米; TR / TE，十五分之五百MS。 </li><li>后成像安装完成后，同步录音和fMRI就可以开始。图2显示了具有代表性的计划免疫图像和成像过程中的原始录音。梯度的快速切换，在饱和的录音，坚持每个扫描周期只一小部分（500分之22MS）的图像采集结果。图像采集后，电信号returNS与非饱和振荡的形式（见图3）基线。可能在静止状态（在这项研究表明），或在刺激进行组合功能磁共振成像和记录。对于刺激研究，成像参数为静止状态的研究相同的前爪电刺激，提供使用9赫兹，1〜4 mA的电流。老鼠安乐死后，最终的扫描。 </li></ol> 3。同时成像和录音后，必须预先的数据处理前最后的分析。 <ol><li>我们首先从拆除的电生理记录（见图3）梯度构件。 <ol><li>由于扫描噪声结构可以提取平均〜500毫秒（TR）的部分，每一个对应的两个连续的功能磁共振图像之间的时间间隔。 </li><li>减去平均噪声结构从原来的录音。这种方法只纠正不饱和录音段。 </li><li>在图像采集相应的渐变交替每个饱和段是一条线，这之间的时间点之前和梯度诱导饱和后的时间点时更换。 </li></ol></li><li>当地外地潜力（LFPs）降噪录音，然后再转换为通电时间的课程，这将有相同的时间分辨率的fMRI时间当然。一个2秒斌内的平均功率是用于计算的最低频率（三角洲带，1〜4赫兹），THETA频段（4〜8赫兹）的频率为1秒斌，斌的〜0.5秒之间邻国更高的频率（&gt; 8赫兹，阿尔法伽玛乐队）的饱和信号。所有频段的滑动窗口被移动了0.5秒递增，匹配的功能磁共振成像数据的TR。 </li><li>图像数据，执行标准功能磁共振成像预处理，包括头部运动校正，图像平滑0.5毫米的FWHM，和线性漂移去除。 </li><li> LFP通电时间的课程，并从每个体素成像数据的时间当然之间进行交叉相关分析。不同的时间滞后允许随时间变化的相关性检查（见图4）。 </li></ol>代表性的成果： 作为一个例子，这种技术可用于调查自发的神经活动和大胆的波动之间的关系。图4显示了LFP权力和BOLD信号在-2.5和9.5小号从一个老鼠之间的时间滞后之间的相关地图。低频大胆的波动从皮质电极尖端附近地区（&lt;0.1赫兹）是与LFP功率变化（&lt;0.1赫兹）的相关延迟了2〜6秒。 <img src="/files/ftp_upload/1901/1901fig1.jpg" alt="图1" /> 图1。电极植入与表面线圈成像区域示意图配置。 <img src="/files/ftp_upload/1901/1901fig2.jpg" alt="图2" /> 图2代表日冕计划免疫的形象，包括电极提示，显示在左侧面板。右侧的面板显示之前和期间成像原料的电生理记录。 <img src="/files/ftp_upload/1901/1901fig3.jpg" alt="图3" /> 图3。放大在一个扫描周期，可以看出，在成像的工件（绿色），可从原始录音删除（蓝色）。降噪课程（红色），用于进一步的分析。 <img src="/files/ftp_upload/1901/1901fig4.jpg" alt="图4" /> 图4。日冕的地图，从一个电极的力量自发三角洲乐队活动在时间和休息状态的BOLD信号之间的相关性（从一个典型的大鼠）滞后于从-2.5到9.5秒最大的相关性是在双边的SI观察，在大约4〜5小号在异氟醚麻醉大鼠。彩条代表皮尔逊河

<ol>
	<li>Gruetter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic,+localized+in+vivo+adjustment+of+all+first-+and+second-order+shim+coils.">Automatic, localized in vivo adjustment of all first- and second-order shim coils.</a> Magn Reson Med. 29 (6), 804-804 (1993).</li><li>Logothetis, N. K., Pauls, J., Augath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurophysiological+investigation+of+the+basis+of+the+fMRI+signal.">Neurophysiological investigation of the basis of the fMRI signal.</a> Nature. 412 (6843), 150-150 (2001).</li><li>Nir, Y., Fisch, L., Mukamel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+between+neuronal+firing+rate,+gamma+LFP,+and+BOLD+fMRI+is+related+to+interneuronal+correlations.">Coupling between neuronal firing rate, gamma LFP, and BOLD fMRI is related to interneuronal correlations.</a> Curr Biol. 17 (15), 1275-1275 (2007).</li></ol>

电生理记录的BOLD功能磁共振成像技术分别发达。然而，同时录制和成像是具有挑战性，由于相互干扰的两 ​​种模式2。在这里，我们提供了一个可能的解决方案结合的啮齿类动物实验。电极植入的修改后的方法，最大限度地减少图像质量的影响，电气录音的工件去除是必要的，以消除噪声引起的图像采集。同时成像和记录在啮齿类动物中，将提供一个自发的神经活动和BOLD信号之间的耦?...

simultaneous fmri and electrophysiology in the rodent brain

要检查的血氧水平依赖（BOLD）的磁共振成像（MRI）信号的神经基础，我们已经开发出一种啮齿动物模型中，功能性磁共振成像数据，并<em&gt;在体内</em&gt; intracortical记录可以同时执行。 MRI和电气录音的结合是技术上具有挑战性，因为用于拍摄的电极扭曲的MRI图像和MRI的收购引起的电气录音中的噪音。为了尽量减少相互干扰的两种模式，分别用玻璃微电极，而不是金属和噪声去除算法实施的电生理数据。在我们的研究中，两个微电极分别植入双侧初级躯体感觉皮层（SI）的大鼠和固定到位。被选定为功能性磁共振成像冠状切片覆盖电极提示。电极轴和固定的位置不包括在图像切片，以避免成像伪影。去除头皮被替换用牙膏降低易感性不匹配，并防止图像吉布斯振铃文物。在快速转换的磁场在图像采集电气录音引起的工件结构特点，平均每个运行的扫描所有周期。成像过程中的噪音结构，然后从原始录音中扣除。降噪时间课程，然后用fMRI数据的进一步分析相结合。作为一个例子，同时采集用于确定自发的功能磁共振成像大胆的信号和带限intracortical电活动之间的关系。同时在啮齿类动物中的功能磁共振成像和电生理记录将在神经科学中的许多令人兴奋的应用，除了阐明的fMRI BOLD信号和神经元的活动之间的关系提供了一个平台。

Watch this Scientific Journal Video about Simultaneous fMRI and Electrophysiology in the Rodent Brain at JoVE.com

Simultaneous fMRI and Electrophysiology in the Rodent Brain

我们已经开发出一种方法同步功能磁共振成像和电生理记录在啮齿类动物的大脑的神经活动和血氧水平依赖（BOLD）的MRI信号之间的关系进行调查，提供了一个平台。

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 ...

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Neuroscience

13.3K 次观看.  Emory University. 我们已经开发出一种方法同步功能磁共振成像和电生理记录在啮齿类动物的大脑的神经活动和血氧水平依赖（BOLD）的MRI信号之间的关系进行调查，提供了一个平台。

视频: Simultaneous fMRI and Electrophysiology in the Rodent Brain

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

同时神经刺激，并记录在水迷宫的设计和构建一个具有成本效益探头

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

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 ...

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 ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

多鼠脑的同时 Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

量化龙脑中的亚细胞泛基蛋白-蛋白酶体活性

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...