Name: a large lateral craniotomy procedure for mesoscale wide-field optical imaging of brain activity
Uploaded: 2017-05-07T13:00:00
Description: Watch this Scientific Journal Video about A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity at JoVE.com

这项工作是由加拿大自然科学和工程研究理事会（NSERC）发现格兰特＃40352支持，校园艾伯塔省创新程序委员会主席，阿尔伯塔省老年痴呆症研究计划，以MHM，并NSERC CREATE在BIF博士生奖学金和AIHS研究生奖学金到MK。我们感谢浦明·沃对这个协议的发展和外科培训，并贝·米尔扎·阿加和狄鞘的养殖。

<html>下面的协议如下莱斯布里奇动物护理委员会（ACC）的指导方针的大学，并根据加拿大动物保护协会（CCAC）的标准进行的。 1.准备<ol><li>对于长期研究阶段，高压灭菌所有打开的手术用品，并确保不育是保持整个手术。如果需要多次手术，手术之间的反应釜。 </li><li>确保有充足的缓冲脑的手头上（至少50 mL）中。该溶液是由134 mM氯化钠，5.4 mM的钾，氯化镁1mM的六水合物，...

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dye+imaging+reveals+dynamic+spatiotemporal+properties+of+cortical+activity+after+spontaneous+muscle+twitches+in+the+newborn+rat.">Voltage-sensitive dye imaging reveals dynamic spatiotemporal properties of cortical activity after spontaneous muscle twitches in the newborn rat.</a> J Neurosci. 32 (32), 10982-10994 (2012).</li><li>Sweetnam, D., 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=Diabetes+impairs+cortical+plasticity+and+functional+recovery+following+ischemic+stroke.">Diabetes impairs cortical plasticity and functional recovery following ischemic stroke.</a> J Neurosci. 32 (15), 5132-5143 (2012).</li><li>Yin, Y. Q., 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=In+vivo+field+recordings+effectively+monitor+the+mouse+cortex+and+hippocampus+under+isoflurane+anesthesia.">In vivo field recordings effectively monitor the mouse cortex and hippocampus under isoflurane anesthesia.</a> Neural Regeneration Research. 11 (12), 1951-1955 (2016).</li><li>Sharp, P. 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=Comparison+of+stimulus-evoked+cerebral+hemodynamics+in+the+awake+mouse+and+under+a+novel+anesthetic+regime.">Comparison of stimulus-evoked cerebral hemodynamics in the awake mouse and under a novel anesthetic regime.</a> Scientific Reports. 5, 12621 (2015).</li><li>Kyweriga, M., Mohajerani, M. H., Kianianmomeni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics:+Methods+and+Protocols.">Optogenetics: Methods and Protocols.</a> Methods in Molecular Biology. 1408, 251-265 (2016).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging in neuroscience and development : a laboratory manual. , (2005).</li><li>Vanni, M. P., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscale+transcranial+spontaneous+activity+mapping+in+GCaMP3+transgenic+mice+reveals+extensive+reciprocal+connections+between+areas+of+somatomotor+cortex.">Mesoscale transcranial spontaneous activity mapping in GCaMP3 transgenic mice reveals extensive reciprocal connections between areas of somatomotor cortex.</a> J Neurosci. 34 (48), 15931-15946 (2014).</li><li>Xie, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+High-Frequency+Mesoscale+Intracortical+Maps+Using+the+Genetically+Encoded+Glutamate+Sensor+iGluSnFR.">Resolution of High-Frequency Mesoscale Intracortical Maps Using the Genetically Encoded Glutamate Sensor iGluSnFR.</a> J Neurosci. 36 (4), 1261-1272 (2016).</li><li>Chan, A. W., Mohajerani, M. H., LeDue, J. M., Wang, Y. T., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscale+infraslow+spontaneous+membrane+potential+fluctuations+recapitulate+high-frequency+activity+cortical+motifs.">Mesoscale infraslow spontaneous membrane potential fluctuations recapitulate high-frequency activity cortical motifs.</a> Nat Commun. 6, 7738 (2015).</li><li>Lim, D. H., 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=In+vivo+Large-Scale+Cortical+Mapping+Using+Channelrhodopsin-2+Stimulation+in+Transgenic+Mice+Reveals+Asymmetric+and+Reciprocal+Relationships+between+Cortical+Areas.">In vivo Large-Scale Cortical Mapping Using Channelrhodopsin-2 Stimulation in Transgenic Mice Reveals Asymmetric and Reciprocal Relationships between Cortical Areas.</a> Front Neural Circuits. 6, (2012).</li><li>Ferezou, I., 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=Spatiotemporal+dynamics+of+cortical+sensorimotor+integration+in+behaving+mice.">Spatiotemporal dynamics of cortical sensorimotor integration in behaving mice.</a> Neuron. 56 (5), 907-923 (2007).</li><li>Mohajerani, M. H., Aminoltejari, K., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mini-strokes+produce+changes+in+interhemispheric+sensory+signal+processing+that+are+indicative+of+disinhibition+within+minutes.">Targeted mini-strokes produce changes in interhemispheric sensory signal processing that are indicative of disinhibition within minutes.</a> Proc Natl Acad Sci U S A. 108 (22), E183-E191 (2011).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+for+intersectional+targeting+of+neural+sensors+and+effectors+with+high+specificity+and+performance.">Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.</a> Neuron. 85 (5), 942-958 (2015).</li></ol>

这对于大颅窗口创新协议使得能够在大脑皮质的时间和顶区同时成像。与光学成像结合，它可以帮助自发的和刺激诱发的活动中来揭示皮层区域内的神经动力学。这种膨胀的开颅手术还公开了一个大的扩展皮质脉管系统的网络，包括大脑中动脉（MCA）的近端，从而使在血流和血管外侧缺血性模型的直接操纵的体内成像。这种技术将是伟大的使用表达电压和钙指标蛋白23</...

开颅是使用神经科学家以显示脑的一部分的标准程序。由于电的曙光，开颅手术已经允许在神经科学领域前所未有的突破。与电极大脑皮层的密集映射导致基于这些地图的实验测试假说和理论。我们最近进入其中开颅正用于皮质血流1，2，3和神经血管结构4的体内成像，使曝光区5，6，7内皮层活动的实时可视化的新时代。虽然许多研究使用与开颅体内光学成像技术相结合，研究结构和皮层神经元，神经胶质的功能，和COR蒂卡尔脉管系统8， 图9，进一步调查由暴露皮质的小区域限定（但参阅10）。 该协议的目的是提供一种用于产生大的横向开颅，从中线暴露大脑皮层到鳞骨，并且延伸超出前囟和lambda提供的方法。这种大的开颅使得关联皮层的同时观看（脾后，扣，和顶叶），原发性和继发性马达，体感，视觉和听觉皮层。这种方法已经被预先加上电压敏感染料成像（VSDI）调查皮层区域如何多个自发的和刺激诱发的皮质活动5，11，12中彼此交互。此过程中最具挑战性的方面包括定位头动物的，固定所述头板，和避免出血，同时从顶骨分离颞肌。护理也必须在钻井和头骨移除过程作为头骨曲线以倾斜的角度拍摄。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Heating Pad&#160;</td>
			<td>FHC</td>
			<td>40-90-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps&#160;</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>2 or more pairs are recommended</td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00, 15000-10</td>
			<td>1 pair should be designated for dura removal&#160;</td>
		</tr>
		<tr>
			<td>Jet tooth shade powder</td>
			<td>LANG Dental</td>
			<td>Jet Tooth Shade Powder</td>
			<td>to be mixed with the Jet Liquid</td>
		</tr>
		<tr>
			<td>Jet tooth shade liquid</td>
			<td>LANG Dental</td>
			<td>Jet Tooth Shade Liquid</td>
			<td>to be mixed wihth the Jet Powder&#160;</td>
		</tr>
		<tr>
			<td>Drill Heads - Carbide Burs FG 1/4 389</td>
			<td>Midwest Dental</td>
			<td>385201</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Powder</td>
			<td>Sigma-Aldrich</td>
			<td>A9793</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Sinclair Dental Canada</td>
			<td>Pfizer Gelfoam</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>124125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine 2% Epinephrine</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>125299</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethazone 5 mg/mL</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>125231</td>
			<td></td>
		</tr>
		<tr>
			<td>Butyl cyanoacrylate glue (VetBond)</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>12612</td>
			<td></td>
		</tr>
	</tbody>
</table>

a large lateral craniotomy procedure for mesoscale wide-field optical imaging of brain activity

开颅是一种常用执行程序以暴露脑用于体内实验。在小鼠研究，大多数实验室利用小开颅，一般为3毫米×3毫米。此协议引入用于产生基本上更大7毫米×6mm的颅窗露出最一个大脑半球的在小鼠颞和顶叶皮层的方法（ 例如，囟2.5 - 4.5毫米，侧0 - 6毫米）。为了执行该手术，头必须倾斜大约30°，并且许多颞肌的必须缩回。由于大量的骨质去除，这种方法仅用于急性实验在整个手术和麻醉实验动物。 这种创新的大的横向颅窗口的主要优点是提供了皮质的两个内侧和外侧的区域同时访问。这个大单边颅窗口可用于研究细胞之间的神经动力学，以及通过组合多电极电生理记录，神经元活动（ 例如，内部或外部成像），和光遗传学刺激的成像作为不同的皮层区之间。此外，这种大开颅手术也暴露出皮层血管大面积的，允许横向皮质血管的直接操作。

<html>为了研究在单个半球内皮层区之间的相互作用，我们使用了跨越矢状窦和5延伸出的大的开颅 - 6毫米横向。此颅窗口包括初级（马达，体感，视觉，听觉），仲（电机，视觉）和关联（脾后，扣，顶叶协会）右脑半球（ 图3A）的皮层。对于这项工作，我们使用电压敏感染料（VSD）成像，这反映在膜电位3变化。该协议也将是其它?...

Watch this Scientific Journal Video about A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity at JoVE.com

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

该协议提出了在小鼠大脑皮质的时间和顶叶区域产生大单方面开颅的方法。这对于实时成像在皮质半球的一个广阔的区域是特别有用的。

大脑侧向颅骨切开术大脑活动的中尺度广角光学成像

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

The overall goal of this large lateral craniotomy is to perform real time imaging over an expansive area of a cortical hemisphere. The main advantage of this technique is that large areas of the cortex are exposed for observation and can be used in conjunction with technologies such as optogenetics. This technique can provide insights into characterizing the neural dynamics between cells as well as that between different brain circuits at different temporal and spatial resolutions.Visual demonstration of this method is critical due to the increased risk of hemmorage and cortical damage since the dura is often attached to brain surface and blood vessels. This method can be applied to study the neurobiological mechanisms underlying brain circuit development and its changes throughout life in response to new experiences or pathologies such as Alzheimer's Disease or stroke. To begin this procedure, autoclave all the surgical supplies and ensure that sterility is maintained throughout the surgery.Ensure there is enough brain buffer for the surgery. Next, transfer an anesthetized mouse to the head holder setup and place it on a thermo-regulating heating pad set to 37 degrees Celsius. Secure the mouse's upper teeth in a teeth holder.Then rotate its head towards the left approximately 30 degrees to expose the right lateral side of the head. Subsequently, secure the mouse's head with the blunt end of the ear bars. Then apply a thalmic ointment to prevent corneal drying.To reduce cerebral edema, inject it with dexamethasone at four milligrams per kilogram intermuscularly. Wipe the skin over the surgical area with cotton swabs dipped in 4%chlorhexidine three times and then with 70%alcohol three times. Inject lidocaine subcutaneously over the craniotomy site.Under a dissecting microscope, lift up the skin at one millimeter left of the midline just behind the ear with forceps and make a small horizontal incision with surgical scissors. Make a five to six millimeter lateral cut towards the right ear and then cut towards the rostral end of the head. At the initial incision point, insert the scissors and cut 10 millimeters rostrally.After that, cut the skin around the right ear and near the right eye to expose the right side of the skull and temporal muscle. Ensure that the widest part of the exposed area is at least seven millimeters. Then trim the skin further if the surgical area needs to be extended.Using a cotton swab, rub the surface of the skull in a circular motion to remove the periosteum from the skull. Ensure that no periosteum remains and that the skull is completely dry, which is critical for successful attachment of the head plate. Next, using spring scissors and forceps, separate the temporal muscle from the skull.Cut and retract the muscle laterally until it reaches the squamosal bone. Take extreme care not to damage the superficial temporal vein that runs along the level of the squamosal bone near the eye, otherwise hemorrhage might occur. Control bleeding with gel foam pre-soaked in brain buffer.For serious hemorrhage, use a heat cauterizer. Drop butyl cyanoacrylate glue onto bleeding sites. To begin this procedure, first mark bregma to orient the underlying brain regions, apply ethyl cyanoacrylate glue around the bottom edge of the head plate and glue the head plate over the craniotomy area.After that, fill the opening space between the skull and the head plate with dental cement, leaving only the craniotomy area exposed. Then wait for the dental cement to dry and harden. Subsequently, outline the cranial window region by lightly scoring the surface of the skull with the dental drill.Gently trace the drill along the original scoring to deepen it, ensuring that the drill does not penetrate through the skull into the brain. In the meantime, switch between drilling and dabbing the skull's surface with moistened rolled tissues every few minutes. This very challenging start requires surgeons to drill through the aged skull to free it for removal without damaging the underlying cortex.Slow is loose, smooth is fast. During drilling, periodically check for buckling of the skull by gently pressing on it with forceps or the non-moving drill bit. When the bone begins the buckle, stop drilling and immerse the entire window in brain buffer.Wait for at least five minutes before skull removal to soften the bone and to reduce the chance of the dura sticking to the bone, making the skull removal process easier. Next, perform the skull removal process beginning at the anterior edge. Gently pry the loose skull from the dura using forceps.Once the bone is loose and floating on the dura, firmly grasp it with forceps and lift it away from the dura. Ensure the bone never penetrates into the brain. Use forceps and spring scissors to gently tear and cut away the dura.Care must be taken to ensure the brain parenchyma and blood vessels are not damaged. Continue until all dura is removed from the cranial window site. When performed correctly, the brain will appear to be very clean with distinct blood vessels and no blemishes.Spray the coverglass with 70%ethanol and use an air canister to gently dry. Ensure the glass is completely clean with no spots or dust present. Next, prepare 1.3%agarose by heating 200 milligrams of agarose powder dissolved in 15 milliliters of brain buffer.Place a thermometer in the hot agarose and cool the agarose down to just above solidifying temperature at about 40 degrees Celsius. Cooling can be expedited by running cold water over the tube, taking care not to allow any water to enter the agar. Gently stir with the thermometer while monitoring its temperature.Immediately before applying the agar, remove the brain buffer from the cranial well. Ensure no bubbles or particles are present as these will interfere with imaging. Now draw up the agarose with a transfer pipe head and drop the agarose directly on the brain.Then quickly place the coverslip over the surface and fasten the coverslip with the agarose drops on the corners. Shown here is a photo micrograph of the wide unilateral craniotomy with bregma indicated by a white circle in each image. Patterns of cortical activation are shown in a mouse anesthetized with 0.5%isoflurane after stimulation of the contralateral whisker, auditory stimulation, contralateral forelimb stimulation, contralateral hindlimb stimulation and visual stimulation of the contralateral eye with a light-emitting diode.There was midline activation from all forms of sensory stimulation at 10 to 25 milliseconds after primary sensory cortex activation. Once mastered, this technique can be completed in three to four hours. When attempting this procedure, it's important to remember to keep the blood loss to minimum and to ensure the cortex is not damaged.Using this procedure, other imaging techniques such as optical imaging can help to answer important questions such as how neural dynamics are influenced by spontaneous activity in the normal and diseased brain. Following its development, this technique helped to pave the way for researchers in the field of systems neuroscience to explore interactions and neural dynamics between widely spaced cortical regions in mice. This video shows you how to perform a large lateral craniotomy in the mouse with minimal blood loss and cortical damage, enabling you to collect the necessary data to answer your most pressing scientific questions.

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State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

依赖国家对TMS的：一看动机幻视行为

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

科研

神经科学

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

Real-Time fMRI Brain Mapping in Animals

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 ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

斑马鱼游泳期间后横向线阿弗伦特神经元的活动

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

体内 使用大颅窗的小鼠宽场和双光子钙成像

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Recording Brain Activity with Ear-Electroencephalography

用耳脑电图记录大脑活动

The c-grid (ear-electroencephalography, sold under the name cEEGrid) is an unobtrusive and comfortable electrode array that can be used for investigating ...