资金来源：这项工作是由基金会资助贝尔塔雷利（DJL），一个MED-EL补助（DJL），以及健康教育资助DC01089国家研究院​​（MCB）的支持。

注：所有的实验程序按照马萨诸塞州眼的动物护理和使用委员会及耳科医院和哈佛医学院，这遵​​循国家动物保健指南，包括公共卫生服务政策上的人文关怀和使用实验动物，在执行ILAR指南，以及动物福利法。上市实验步骤如下左DCN的细节曝光。使用无菌器械，同时执行的生存术。 1.主开颅手术和背耳蜗核曝光<ol><li>麻醉<ol><li>通过腹腔内给药麻醉小鼠，年龄8-12周，体重18-24克，用甲苯噻嗪20mg / kg的氯胺酮和100毫克/公斤。确定合适的麻醉监测心脏，呼吸率，以及脚趾捏撤回反射。对眼睛的地方兽医软膏，以防止干燥，而在麻醉下。一旦充分麻醉下，头发剃覆吨他头皮以提供无障碍地访问手术部位。 </li></ol></li><li>手术定位<ol><li>将鼠标牢固在小动物立体定位架，由一吻夹具保持在适当位置。 </li><li>确保吻钳位足够松，以便有足够的呼吸，但不够紧完全固定在鼠标的头部。如果鼠标头是松散的，动物是可能的过程的开颅部分期间掉出的头支架。 </li><li>放置在标准的方式，这允许心脏速率监测听觉脑干植入电极。呼吸频率应该由可视化监控。需要注意的是正常的心脏速率和呼吸速率会根据鼠标的年龄而变化。 </li><li>温度计放置和euthermia确保通过放置在恒温加热毯。 </li></ol></li><li>切开皮肤及颅骨interparietal和枕骨骨曝光<ol><li>下一个microscOPE，使透过皮肤的垂直切口处开始的中线，耳廓直接之间，并延伸到后头部的尾部。下面正中切开皮肤，横向位移皮肤。可视覆盖颅骨的左后侧方面的肌肉。 </li><li>肌肉附件离断取下的肌肉覆左顶叶，interparietal和颅骨枕骨骨各自的骨头用手术刀或虹膜剪刀。观察一个小的程度以及切割边缘的肌肉出血。通过用棉签10-15秒温和的压力减少了。 </li></ol></li><li>开颅覆耳蜗核<ol><li>确定相关缝合线，包括矢状面和lambda缝合线（ 图1，左图）。 </li><li>用咬骨钳，作开颅手术在interparietal骨，中线左侧，〜2毫米尾鳍在lambda缝合线。这个区域覆盖了DCN。 </li><li>跟随着克开颅手术，观察薄层硬膜覆盖在小脑。使用手术刀，删除硬脑膜。 （ 图1，中图）。 注：去除硬脑膜可能导致小程度的出血。使用手术刀刀片去除硬脑膜的这个过程是为了减少小脑抽吸，这将集中在脑干表面和模糊的识别与DCN期间血液损失量。小鼠总血量为〜1.5毫升不应超过&gt;低于15％。如果出血超过这个数额，鼠标应提供与补液。 </li><li>取出凝固的血液覆盖的棉签涂抹小脑。或者，用牙科点到小脑清除血液离开血淌轻轻生理盐水。 </li></ol></li><li>小脑吸<ol><li>使用5法语吸，吸左侧小脑覆DCN的最外侧部分，直到与DCN是相UAL（ 图1，右图 ）。卸下左侧小脑大约1/4到1/3。毗邻DCN的主要地标是上半规管的壶腹。 注：小脑心愿是协议的关键一步。如果它发生在一个单一的企图，而不是与吸多遍，因为这会导致出血是最成功的进行了小脑导演的愿望。以协助抽吸，设置显微镜的焦平面处，CN，这将是略微远端小脑的表面的期望的深度。焦平面设置到CN将改进的可视化，并确保一个鲜明的图​​像。 </li><li>下面愿望，再出血，脑脊液（CSF）集结起来，小脑排量的DCN预期。灌输0.5毫升的无菌盐水的迅速进入开颅以防止血液凝固。然后轻柔涂抹的用牙科点和吸入的组合可以是使用扫清了DCN直接可视化的路径。不要直接接触DCN的表面。 </li></ol></li></ol> 2.压力显微注射的病毒介导的基因转移和手术恢复<ol><li>与DCN是免费的覆盖血液和脑脊液后和清晰可见，用10微升Hamilton注射器在2分钟内使压力显微注射入DCN。 </li><li>用显微操作引入针，直到针尖不再与DCN的表面下可见。 </li><li>为了获得最佳性能，使用33或34号针（使用气密注射器用34号针头）与浅斜面，如45°，以减少钝伤和本地化DCN内注射量，这是一种薄和浅脑干结构（&lt;300微米厚）。 注意：其他注射手段可以基于利用。理想情况下，注射仪器应具有灵活性，允许一些轻微的奔D，如果需要直接针对DCN。另外，作为小鼠轻微移动整个手术过程中，由于呼吸，仪器应是坚固足以承受运动的少量。 </li></ol> 3.手术恢复<ol><li>紧接着注射，重新接近皮肤，让鼠标按标准恢复过程来恢复。其余小脑和疤痕组织将填补引起小脑愿望腔。不断地监测动物，直到足够的意识恢复，保持胸骨斜卧。监测心脏速率，呼吸速率，以及饲料的能力。 注：没有动物已经动过手术返回到与其他动物的笼子，直到完全康复。 </li><li>如果鼠标开始走在圈子手术后（&lt;5％的病例），皮肤切口关闭后通常2-4小时，立即牺牲鼠标，因为它很可能有困难的时候喂食。牛逼他的副作用，可能是由于小脑的愿望。动物应为疼痛，术后进行监测，并适当麻醉品应给予保证根据机构的标准动物的舒适性。抗生素可能是必要的，如果感染的证据。 </li></ol> 4.二次开颅手术和人工耳蜗核曝光<ol><li> 2-4周的愈合和病毒介导的基因转移的潜伏期后，重新麻醉与基因转移的类型预先注射小鼠，并重复步骤1.1-1.5孵育时间而变化。 </li><li>由镊子，解剖刀，和咬骨钳的组合去除瘢痕组织在开颅站点。 </li><li>可视化开颅后，确定剩余的小脑和疤痕组织覆盖在DCN的组合。使用5弗伦奇吸入到吸覆小脑/疤痕组织（类似于前述小脑抽吸）。 </li><li>下面的心愿，期待再出血，脑脊液Build镜头，和小脑的其余位。快速引入盐水进开颅以防止血液凝固。用温和的牙齿涂抹点和吸力的组合来清除的DCN直接可视化的路径。 </li><li>观察到DCN的表面上。执行光遗传学基础生理学实验为DCN现在访问。引入光刺激，例如用光纤（ 图2），以驱动一个光遗传学系的实验。 </li></ol> 5.组织学<ol><li>下面的实验结论，安乐死鼠标氯胺酮的过量。灌注小鼠生理盐水后跟4％多聚甲醛。 </li><li>提取的头骨和后固定2小时脑干。 Cryoprotect在30％蔗糖的脑干中24-48小时。使用一节使用60微米的部分标准的低温​​恒温器脑干。 </li></ol>

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作者什么都没有透露。

本文介绍了DCN在操纵中枢听觉系统的小鼠模型直接可视化的技术。直接可视化的概括方法提供了主要的替代显著的优势，这是立体的方法。为主，DCN的直接可视化允许对脑干部位的立即确认，而立体定向的方法不能提供直接可视化。在于必要延长潜伏期，如在病毒介导的基因转移的情况下的实验中，存在用于低感染效率的潜力，如果注射液“射门”时，目标位置。另外，直接可视化的方法允许用?...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

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			<td height="42" style="height:42px;width:197px;">Name of the Material / Equipment</td>
			<td style="width:227px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
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			<td height="21" style="height:21px;width:197px;">Stereotaxic holder</td>
			<td style="width:227px;">Stoelting</td>
			<td>51500</td>
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			<td height="42" rowspan="2" style="height:42px;width:197px;">Homeothermic blanket</td>
			<td rowspan="2" style="width:227px;">Harvard</td>
			<td rowspan="2">507214</td>
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			<td height="21" style="height:21px;width:197px;">Scalpel blade #11</td>
			<td style="width:227px;">Fine Surgical Tools</td>
			<td>10011-00</td>
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		<tr height="21">
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		<tr height="30">
			<td height="51" rowspan="2" style="height:52px;width:197px;">10 &#181;l Hamilton syringe</td>
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		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:197px;">34 gauge, needle</td>
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		<tr height="21">
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		<tr height="21">
			<td height="21" style="height:21px;">Rongeurs</td>
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direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

调查使用病毒介导的基因转移到阻止或逆转听力丧失在很大程度上被转移到外周听觉系统。很少有研究探讨基因转移到中央听觉系统。脑干的背侧耳蜗核（DCN），它包含了听觉通路的二阶神经元，是用于基因转移的潜在位点。在这个协议中，对于鼠DCN经由后颅窝的方法直接和最大曝光技术被证实。这种方法允许对急性或存活手术。继DCN的直接可视化，一台主机的实验是可能的，包括注射视蛋白进入耳蜗核和随后刺激的由光纤耦合到蓝色光的激光。其他神经生理学实验，如电刺激和神经注射器描也是可行的。visualiza的水平化和刺激的持续时间可达到使这种方法适用于广泛的实验。

部分小脑吸入演示进入耳蜗核 后的皮肤和肌肉覆盖在颅骨被除去，颅骨表面的地标，例如冠状和LAMDA缝合线，表明开颅的大致定位。下面开颅咬骨钳，小脑可视化。小脑的小部分仔细吸出表明该CN，然后可将其注入（ 图1）的可视化。 二级耳蜗核暴露的刺激与蓝光激光器 继DCN，与视蛋白的基因转移，和潜伏...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

Research

JoVE Journal

Neuroscience

9.1K 次观看.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

视频: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS：使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

科研

神经科学

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS的设备进行美国FDA批准用于治疗抑郁症的议定书“

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

经颅直接电流刺激电极定位和蒙太奇

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

在癌神经侵袭的体外模型：背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...