这项研究是由美国国家药物滥用研究所的支持给予DA 006886（MOW）和DA 032270（DJB）。

悉心照顾，必须采取保持无菌条件下（如在指南中描述的实验动物9的护理和使用），同时准备和进行以下步骤。以下协议符合本指南实验动物的护理和使用，并已被批准的机构动物护理和使用委员会，罗格斯大学。据估计，在后续的步骤将需要3-6小时来完成。 植入微丝阵列： <ol><li>在麻醉状态下的动物的地方使用50毫克/千克戊巴比妥钠（IP）和管理硝酸甲基阿托品10毫克/千克（IP;格隆可取代）和0.25毫克青霉素（30万单位/毫升IM），以维持呼吸功能，防止感染元。 注意：使用此植入手术的3-6小时的长度，戊巴比妥钠的使用，因为它具有成本效益和人的接触限值为麻醉剂（如可能与长期使用麻醉气体的发生），同时还提供长效麻醉。其他麻醉剂的​​替代是可以接受的。 </li><li>确认麻醉已经使用了尾巴捏在进行测试之前生效。 </li><li>必要时，交替给予盐酸氯胺酮（60毫克/千克IP）和戊巴比妥钠（5-10毫克/千克IP）注射，以维持整个手术麻醉。 </li><li>使用＃22手术刀刀片刮头皮。 </li><li>消毒头皮剃光用聚维酮碘。 </li><li>给予布比卡因皮下注射（〜1毫克/千克SC超过4注射部位扩散）在本地麻醉头皮。允许5-10分钟的局部麻醉生效。 </li><li>将眼科润滑剂过来的目光，以麻醉过程中保持水分。 </li><li>保护动物进入耳内的酒吧和一个立体定位仪的鼻夹。 </li><li>在韦伯斯特使用视觉地标（ 如单杠eotaxic帧），以近似水平的动物的头。这一步只是为了近似水平的头骨。 </li><li>使沿着用＃11解剖刀刀片安装在刀架的头皮中线切口。该切口必须仅从耳朵后面延伸到鼻骨的后部。 </li><li>使用夹层铲，清除所有剩余组织的头骨，直到两个侧面头骨脊和后脊头骨已经达到。 </li><li>拉回来用了一些止血的（10倍）切口周围皮肤。 </li><li>清洁的任何血骷髅，并允许它干。如果发生任何剩余的出血，终止与一个小工具烧灼残余出血。确保颅骨保持清洁和干燥可以在后续步骤中使用的牙科用丙烯酸系结到头骨永久。 </li><li>标记前囟和lambda（ 图2D）通过内插颅骨缝的交点。解剖麦克风roscope要准确标记这些位置。 </li><li>附上一个小尖项目（ 如针或牙科钻头），以一个立体的手臂，并降低它来确定背/腹（DV）前囟和lambda坐标。 </li><li>调整鼻夹，直到DV对方的坐标为前囟和lambda是在100微米（0.1毫米）。 </li><li>一旦整平，记录前/后（AP），内侧/外侧（ML）和前囟的DV坐标随着鼻部夹钳的位置。仔细检查坐标的准确性，为手术的其余部分取决于这些坐标的精度。 </li><li>使用所测量的坐标来计算ML和AP坐标的四个角落“颅骨窗”相对于前囟（ 即开颅; 图1）。颅骨窗口是一个精确定位的矩形，通过它的微丝阵列将成为过去。 </li><li>使用计算出的坐标，以纪念用尖头立体依恋和钢笔墨水的头骨颅骨窗口坐标。以获得精确的标记，只适用于少量的油墨来使用的棉签的划线工具的前端。 </li><li>通过在一系列小的层去除骨钻出来的颅骨窗。 <ol><li>开始钻窗口，其中的标记已经被放置在标记角部。 </li><li>接下来，连接边角，并概述窗口。 </li><li>最后，明确了下到硬膜的深度轮廓内的区域。孔必须更宽（ 即斜角）颅骨的表面层下面，以确保该窗口保持适当的宽度从顶部向底部。 </li></ol></li><li>使用microforceps小心取出颅骨窗内的任何剩余的骨片，碎片，或硬膜。这个步骤是非常重要的，因为材料的这些位可以降低在危及该阵列的完整性。一旦清除，必须保持日Ë窗口湿润抑菌盐水对手术的其余部分。 </li><li>将标记上的骷髅头骨5螺丝和1地线（ 图1）。这些位置将取决于对象的大脑区域而有所不同。该探头将是最安全的，如果一个螺钉被放置在每个5头骨的骨头。在不会与微丝阵列安置干涉位置放置两个头骨螺钉和接地线。 </li><li>钻孔的颅骨螺钉和固定的螺丝。螺丝必须降低，直至只有3-4线程显示（或者，如果使用的螺丝除了那些推荐以外，深足穿越头骨的厚度，以促进数组稳定性，但不宜过深，从而损坏皮质）。放置后清洁螺丝的螺纹。 </li><li>钻探地线一个洞。降低线缓慢（超过1-2分钟）至目标坐标的DV，用牙科丙烯酸固定电线。 </li><li>加入丙烯酸线程各地的头骨螺钉。丙烯酸干燥之前，请取出流远离接地线或颅骨螺钉任何多余的水泥。允许15分钟的牙科丙烯酸干燥/硬化。 </li><li>附加阵列的立体手臂。水平阵列和方向，使会正视穿过颅骨窗口的范围。 </li><li>置于盐水中的颅骨窗，以便它是与颅骨的水平。降低阵列，直到它会在盐水酒窝。该坐标被用作阵列头骨的水平。使用这个值来计算数组的最后DV坐标。 </li><li>降低阵列缓慢，直到它到达其最终的DV协调。停止降低每1mm至等待几分钟，以便使脑组织，以从凹陷恢复，并在阵列上溶解的聚乙二醇（PEG）的底部部分（PEG是用于暂时保持的电线在其构象，同时降低，并慢慢地溶解在生理盐水中）。 </li><li>当数组为1mm远离目标，降低它更慢，直到最终达到目标。使组织休息一段5分钟前在0.1-0.2毫米在一个时间降低是为了保存组织在靶位点以及近端的突触连接。如果设备是可用的，精度降低阵列可以使用机动操纵器的协助。 </li><li>溶解剩余的PEG和使用牙科丙烯酸水泥到位的微丝。加水泥的多个层，以确保导线是安全的，则允许15-20分钟为水泥，然后再继续硬化。这确保了导线将不会被从它们的最终位置推挤。 </li><li>使用牙科丙烯酸打造动物的探头的剩余部分。使用丙烯酸包住颅骨螺钉，接地线和连接器的微丝。使丙烯酸帽子，在继续操作之前充分硬化。 </li><li>使用可吸收缝合线缝合头皮切口和辖2毫升邻f抑菌液（SC）从手术恢复水化。 </li><li>从耳棒取出动物，并把这个问题在一个干净的区域。手术后的恢复过程中经常观察动物，直到体温调节和运动已经恢复。下面从麻醉中恢复，将动物分为单房为手术后恢复的其余部分。 </li><li>给动物每天术后监测和关怀的日子下面的步骤。恢复从这个过程是最优时是允许的7天或以上。 </li><li>给动物卡洛芬（为5mg/kg）和恩诺沙星每推荐的主治兽医的时间表恢复期间（5-10毫克/千克）或它们的等价物注射。 </li></ol>

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作者没有竞争经济利益披露。

细胞外记录代表一个强大的实验技术，它可以被纳入几乎所有的实验准备在神经科学。已经植入在有组织的阵列线可被跟踪为它们的轴穿过大脑并进入其靶区域（ 图5A）。当在非绝缘微丝尖端创建一个小的，实验后病变从不锈钢线创建一个小的铁矿床，可以使用溶液精确地标记非绝缘微丝尖端的位置（其中，被记录的单单元） 5％亚铁氰化钾和10％的HCl（ 图5A和5B;</s...

电生理记录让科学家研究生物细胞的电特性。在中枢神经系统中，其中电脉冲作为信号传导机制，这些录音是为了解神经功能1-2特别重要的。在单一单元中记录行为的动物，其已被插入到大脑中的微电极能够在一段时间内神经元的产生动作电位的变化进行记录。 虽然许多技术允许一个记录大脑活动，单机电是最精确的方法，允许的分辨率在单个神经元的水平之一。当空间特异性程度高是需要的，微丝可以用于靶向离散子细胞核或brain3内细胞的合奏。单机录音也受益于高时间分辨率的录音是准确的在微秒级。并且， 在体内唤醒录音允许完整电路的相互作用，与传入和传出突起，全身化学和激素的影响，和生理参数的天然环境。神经信号从感觉输入，运动行为，认知过程，神经化学/药理学，或者某种组合。因此，感觉，运动，认知和化学影响的偏析就必须周密的实验，有效的突发事件和控制，可以允许每个上述影响的评估。总而言之，在录音动物行为让实验者观察多个信息源的集成功能电路内，并推导出电路功能更全面的模型。 单单元记录也具有许多缺点的，其中任何实验者应该意识到受损。首先，录音可能难以进行。事实上，日的性质Ë探头放大器和植入微丝，使空间和时间特异性，这些记录也使得录音易受外来电信号（ 即电气“噪声”）的影响。因此，在一个电系统解决问题的能力，就必须电生理原理和装置的发达技术的理解。同样重要的是要注意，在某些情况下，在细胞外记录记录的电信号可代表多个神经信号的总和。此外，单单元活动的目标区域内的人口活动的普遍性通常可以通过细胞异质性的目标区域内的程度的限制（但参阅卡丁4）。例如，电极可能会偏向于记录高振幅输出神经元，以代替其他细胞。单单元记录的可解释性增加通过组合记录与其它技术包括，但不限于，电气（顺或逆向），化学（ 例如，离子电渗疗法或设计师受体）或光遗传学刺激4，临时神经inactivations，感觉检查5，断开的程序，或免疫组织化学3。 在下面，我们将列举必需的材料和步骤，植入大鼠的组织微丝阵列（尽管该协议可以适于在其他物种中使用）的协议。在我们的实验室中使用的固定阵列的过程和风格已被证明可靠的纵向录音，可以在一个月的时间内维持6-8相同的神经元记录的。这使得这个过程非常适用于研究阶段性反应实验的刺激，塑料改变神经反应，或者学习和动机的机制。

<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Table 1. List of Surgical Materials</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisher (MooreBrand)</td>
			<td>19-898-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Swabs</td>
			<td>Fisher (Puritan)</td>
			<td>S304659</td>
			<td></td>
		</tr>
		<tr>
			<td>Nembutal (Pentobarbital)</td>
			<td>Sigma Aldrich</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Methyl Nitrate</td>
			<td>Sigma Aldrich</td>
			<td>A0382</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril (Enrofloxacin)</td>
			<td>Butler Shein (Bayer)</td>
			<td>1040007</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride</td>
			<td>Butler Shein</td>
			<td>SKU# 023061</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine (Povidone-Iodine)</td>
			<td>Fisher (Perdue)</td>
			<td>19-066452&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotax</td>
			<td>Kopf</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Cauterizing Tool</td>
			<td>Stoelting</td>
			<td>59017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Nikon</td>
			<td>SMZ445</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Drill</td>
			<td>Buffalo</td>
			<td>37800</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriostatic Saline</td>
			<td>Bulter Schein</td>
			<td>8973</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewlers Skrews</td>
			<td>Stoelting</td>
			<td>51457</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwire Array</td>
			<td>Microprobes</td>
			<td>Custom (Flexible)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ground Wire</td>
			<td>Omnetics</td>
			<td>Custom Plug</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Acrylic</td>
			<td>Fisher (BAS)</td>
			<td>50-854-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable Sutures</td>
			<td>Fisher (Ethicon)</td>
			<td>NC0258473</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube (Opthalamic Ointment/Lubricant)</td>
			<td>Fisher (Henry Schein)</td>
			<td>008897</td>
			<td></td>
		</tr>
	</tbody>
</table>
<table>
	<tbody>
		<tr>
			<td>Table 2. List of Surgical Instruments</td>
		</tr>
		<tr>
			<td>2x Microforceps</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#160-57</td>
			<td>Multi-use (e.g. clearing debris in skull window)</td>
		</tr>
		<tr>
			<td>2x Forceps</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#160-93</td>
			<td>Multi-use (e.g. tying sutures)</td>
		</tr>
		<tr>
			<td>6x Hemostats</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-1125</td>
			<td>Clamp and open incision</td>
		</tr>
		<tr>
			<td>1x Small scissors</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-411</td>
			<td>Cut sutures after tying</td>
		</tr>
		<tr>
			<td>1x Tissue forceps</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-222</td>
			<td>Holding tissue while suturing</td>
		</tr>
		<tr>
			<td>1x Needle holder</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-1259</td>
			<td>Holding suture needle</td>
		</tr>
		<tr>
			<td>1x Scalpel holder (with #11 blade)</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#105-80 (w/ #105-71 blade)</td>
			<td>Making skull incision</td>
		</tr>
		<tr>
			<td>1x # 22 Scalpel blade</td>
			<td>George Tiemann &#38; Co.</td>
			<td># 160-381</td>
			<td>Shaving scalp</td>
		</tr>
		<tr>
			<td>1x Surgical Spatula</td>
			<td>George Tiemann &#38; Co.</td>
			<td>#160-718</td>
			<td>Scraping skull to clear tissue on skull</td>
		</tr>
		<tr>
			<td>Machine/Jewelers Screws</td>
			<td>Various</td>
			<td>N/A</td>
			<td>0/80 x 1/8&#8221;</td>
		</tr>
	</tbody>
</table>
<table>
	<tbody>
		<tr>
			<td>Table 3. List of Equipment for Recording Electrophysiological Signals</td>
		</tr>
		<tr>
			<td>Microwire Array &#38; Connector</td>
			<td>Micro Probe, Inc. (Gaithersburg, MD)</td>
			<td>&#160;N/A</td>
			<td>Cranially implanted in target recording region. Arrays are customized based on desired wire spacing, length, etc.</td>
		</tr>
		<tr>
			<td>(Part No. Based on array characteristics)</td>
		</tr>
		<tr>
			<td>Unity-Gain Harness/Headstage</td>
			<td>M.B. Turnkey Designs (Hillsborough, NJ)</td>
			<td>Proj 1200</td>
			<td>Initial amplification of neural signal; allows for propagation of small neural signals.</td>
		</tr>
		<tr>
			<td>Commutator (&#38; Optional Fluid Swivel)</td>
			<td>Plastics One, Inc. (Roanoke, VA)</td>
			<td>SL18C</td>
			<td>Allows animals to freely rotate while propagating electrical signal to preamp</td>
		</tr>
		<tr>
			<td>Pre-Amplifier</td>
			<td>M.B. Turnkey Designs (Hillsborough, NJ)</td>
			<td>Proj 1198</td>
			<td>Differentially amplifies neural signals against a reference electrode.</td>
		</tr>
		<tr>
			<td>Filter &#38; Amplifier</td>
			<td>M.B. Turnkey Designs (Hillsborough, NJ)</td>
			<td>Proj 1199</td>
			<td>Band-pass filters and further amplifies the differentially amplified signal.</td>
		</tr>
		<tr>
			<td>Acquisition Computer</td>
			<td>EnGen (Phoenix, AZ)</td>
			<td>N/A (Custom Build)</td>
			<td>Runs software and hardware for behavioral and neural data acquisition.</td>
		</tr>
		<tr>
			<td>A/D Card&#160;</td>
			<td>Data Translation (Marlboro, MA)</td>
			<td>DT-3010</td>
			<td>Digitizes neural signals for computer sampling.</td>
		</tr>
		<tr>
			<td>Digital I/O Card</td>
			<td>Measurement Computing (Norton, MA)</td>
			<td>PCI CTR-05</td>
			<td>Acquires behavioral inputs and outputs</td>
		</tr>
	</tbody>
</table>

a procedure for implanting organized arrays of microwires for single-unit recordings in awake, behaving animals

体内电生理记录在清醒，行为的动物提供了强有力的方法对理解神经信号在单细胞水平。该技术允许实验者在时间和区域研究具体的放电模式，以提供持续的行为相关记录的动作电位。此外，单个单元的记录可以与为了产生神经功能的全面解释了大量的其他技术相结合。在这篇文章中，我们描述了麻醉，准备微丝植入。随后，我们列举了必要的设备和手术步骤，准确地插入微丝数组转换为目标结构。最后，我们简要地描述用于从阵列中的每个单独的电极来记录设备。所述的固定微丝阵列非常适合于慢性注入并允许神经数据的纵向记录在几乎任何行为编写，上。我们讨论了追踪电极轨道进行三角微丝的位置，以及如何以植入微丝结合免疫组化技术，以提高记录结果的解剖特异性。

一个用本实验室记录电生理信号设备的列表可以在表3中可以找到。以下从手术恢复，单单元通过插入一个单位增益探头插入植入连接器记录。此探头通过电缆连接到一个换向器，其能够自由旋转而不在电生理记录符通过使用电滑环。换向器允许受试者在行为，这是该制剂的原理优势之一在录制过程中自由移动。信号，然后通过前置放大器（10倍的增益），用于差分放大信号对所选择?...

Watch this Scientific Journal Video about A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals at JoVE.com

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

植入微丝的组织阵列中单个单元的电生理记录的使用存在许多技术上的挑战。方法来执行这种技术和必要的设备描述。此外，有益利用有组织的微丝阵列由不同的神经次区域具有高空间选择性记录进行了讨论。

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

a-procedure-for-implanting-organized-arrays-microwires-for-single

Research

JoVE Journal

Neuroscience

13.1K 次观看.  Rutgers, the State University of New Jersey. 植入微丝的组织阵列中单个单元的电生理记录的使用存在许多技术上的挑战。方法来执行这种技术和必要的设备描述。此外，有益利用有组织的微丝阵列由不同的神经次区域具有高空间选择性记录进行了讨论。 

视频: A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

电极的制作及植入海兔californica完整的，自由的行为的动物多通道的神经和肌肉的录音

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

科研

神经科学

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

单机在体内</em从大鼠视交叉>录音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined curved surfaces, and images of real-world objects (with and without disparity) in a similar manner as described in the ventral visual stream. In addition, parieto-frontal areas are believed to convert visual object information into appropriate motor outputs, such as the pre-shaping of the hand during grasping. To better characterize object selectivity in the cortical network involved in visuomotor transformations, we provide a battery of tests intended to analyze the visual object selectivity of neurons in parieto-frontal regions.

A detailed protocol to analyze object selectivity of parieto-frontal neurons involved in visuomotor transformations is presented.

用单单元记录法研究猕猴背视流中的对象表示

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined ...

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 ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in 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 ...