Name: in vivo calcium imaging of neuronal ensembles in networks of primary sensory neurons in intact dorsal root ganglia
Uploaded: 2023-02-10T13:20:00
Description: Watch this Scientific Journal Video about In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia Video at JoVE.com

这项工作得到了美国国立卫生研究院拨款R01DE026677和R01DE031477（致YSK），UTHSCSA启动基金（YSK）和德克萨斯大学系统（YSK）的新星奖的支持。

此处描述的所有程序均按照德克萨斯大学圣安东尼奥健康科学中心机构动物护理和使用委员会批准的协议进行。
注意：一旦开始，动物手术（步骤1）和成像（步骤2）必须以连续的方式完成。数据分析（步骤3）可以在以后进行。
1. 手术和固定动物进行右侧 L5 DRG 成像
注意：本研究使用了8周龄或以上的雄性和雌性Pirt-GCaMP3 C...

<ol>
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持续性疼痛存在于多种疾病中，使约8%的人虚弱和/或降低生活质量29。初级感觉神经元检测皮肤上的有害刺激，它们的可塑性有助于持续疼痛8。虽然可以在细胞培养和外植体中研究神经元，但这样做会将它们从正常的生理环境中移除。DRG的手术暴露，然后进行Pirt-GCaMP3 Ca2+成像，允许使用施加在后爪的刺激在正常生理环境中研究初级感觉神经元。这种Ca<s...

初级感觉神经元直接支配皮肤并将体感信息带回中枢神经系统1，2。背根神经节（DRGs）是由10，000-15，000个初级感觉神经元组成的细胞体簇3，4。DRG神经元表现出不同的大小、髓鞘形成水平以及基因和受体表达模式。较小直径的神经元包括疼痛感应神经元，较大直径的神经元通常对非疼痛的机械刺激做出反应5，6。初级感觉神经元的疾病，如损伤、慢性炎症和周围神经病，可以使这些神经元对各种刺激敏感，并导致慢性疼痛、异常性疼痛和疼痛超敏反应7，8。因此，DRG神经元的研究对于理解一般的躯体感觉和许多疼痛和瘙痒疾病都很重要。
体内放电的神经元对躯体感觉至关重要，但直到最近，研究体内完整神经节的工具仅限于相对较少的细胞数量9。在这里，我们描述了一种强大的方法，用于在体内集合中研究群体水平上神经元的动作电位或活动。该方法采用基于细胞质Ca2+动力学的成像。由于细胞质Ca 2+的浓度通常较低，Ca2+灵敏的荧光指示剂是测量细胞活性的良好代理。这些指标允许同时监测小鼠9，10，11，12，13，14，15，16和大鼠17中的数百至数千个初级感觉神经元。本研究中描述的体内Ca2+成像方法可用于直接观察群体对机械，冷，热和化学刺激的反应。
磷酸肌醇结合膜蛋白Pirt在几乎所有（>95%）原代感觉神经元18，19中均高水平表达，可用于驱动Ca2+传感器GCaMP3的表达，以监测体内神经元活性20。在该协议中，描述了使用共聚焦激光扫描显微镜（LSM）在Pirt-GCaMP3小鼠14的右侧腰椎5（L5）DRG中进行体内DRG手术，Ca2 +成像和分析的技术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anased Injection (Xylazine)</td>
			<td>Covetrus, Akorn</td>
			<td>33197</td>
			<td></td>
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			<td>C Epiplan-Apochromat 10x/0.4 DIC</td>
			<td>Cal Zeiss</td>
			<td>422642-9900-000</td>
			<td></td>
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			<td>Cotton Tipped Applicators</td>
			<td>McKesson</td>
			<td>24-106-1S</td>
			<td></td>
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			<td>Curved Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13007-12</td>
			<td></td>
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			<td>DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
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			<td>DC Temperature Controller Heating Pad</td>
			<td>FHC</td>
			<td>40-90-2-05</td>
			<td></td>
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			<td>Dumont Ceramic Coated Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-50</td>
			<td></td>
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			<td>FHC DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluriso (Isoflurane)</td>
			<td>MWI Animal Health, Piramal Group</td>
			<td>501017</td>
			<td></td>
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			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>16221-14</td>
			<td></td>
		</tr>
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			<td>GelFoam</td>
			<td>Pfizer</td>
			<td>09-0353-01</td>
			<td></td>
		</tr>
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			<td>Ketaset (Ketamine)</td>
			<td>Zoetis</td>
			<td>KET-00002R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminescent Green Stage Tape</td>
			<td>JSITON/ Amazon</td>
			<td>B803YW8ZWL</td>
			<td></td>
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		<tr>
			<td>Matrx VIP 3000 Isoflurane Vaporizer</td>
			<td>Midmark</td>
			<td>91305430</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting spring scissors</td>
			<td>Roboz</td>
			<td>RS-5677</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Rectal Thermistor Probe</td>
			<td>FHC</td>
			<td>40-90-5D-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Roboz</td>
			<td>RS-6812</td>
			<td></td>
		</tr>
		<tr>
			<td>Pirt-GCaMP3 C57BL/6J mice</td>
			<td>Johns Hopkins University</td>
			<td>N/A</td>
			<td>Either sex can be imaged equally well. Mice should be at least 8 weeks old due to weak or intermittent Pirt promoter expression in younger mice.</td>
		</tr>
		<tr>
			<td>SMALGO small animal algometer</td>
			<td>Bioseb In vivo Research Instruments</td>
			<td>BIO-SMALGO</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Kopf Model 923-B</td>
			<td>923-B</td>
			<td></td>
		</tr>
		<tr>
			<td>td-Tomato C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>7909</td>
			<td></td>
		</tr>
		<tr>
			<td>Top Plate, 6 in x 10 in</td>
			<td>Newport</td>
			<td>290-TP</td>
			<td></td>
		</tr>
		<tr>
			<td>TrpV1-Cre C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>17769</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 800 confocal microscope</td>
			<td>Cal Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen 2.6 Blue Edition Software</td>
			<td>Cal Zeiss</td>
			<td>Zen (Blue Edition) 2.6</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo calcium imaging of neuronal ensembles in networks of primary sensory neurons in intact dorsal root ganglia

Ca 2+成像可用作细胞活动的代理，包括动作电位和涉及Ca 2+进入细胞质或释放细胞内Ca2+储存的各种信号机制。基于Pirt-GCaMP3的小鼠背根神经节（DRG）初级感觉神经元的Ca2+成像提供了同时测量大量细胞的优势。可以监测多达 1，800 个神经元，允许在体内种群水平的正常生理环境中将神经网络和体感过程作为一个整体进行研究。监测的大量神经元允许检测使用其他方法难以检测的活动模式。刺激可以应用于小鼠后爪，从而可以研究刺激对DRG神经元系综的直接影响。产生 Ca 2+ 瞬变的神经元数量以及 Ca2+ 瞬变的振幅表明对特定感觉模式的敏感性。神经元的直径提供了活化纤维类型的证据（非有害机械纤维与有害疼痛纤维，Aβ，Aδ和C纤维）。表达特定受体的神经元可以用td-Tomato和特异性Cre重组酶以及Pirt-GCaMP进行遗传标记。因此，DRG的Pirt-GCaMP3 Ca2+成像为分析特定的感觉模式和神经元亚型提供了一个强大的工具和模型，在人群水平上作为一个集合来研究疼痛，瘙痒，触摸和其他躯体感觉信号。

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64826/64826fig04.jpg"> 图4：Pirt-GCaMP3小鼠L5背根神经节的代表性图像。 （A，D）显示了Pirt-GCaMP3小鼠L5背根神经节的单帧高分辨率扫描。（乙，东）.在没有刺激的情况下，分别来自图A和图D的15帧Pirt-GCaMP3 L5 DRG神经节的平均强度预测。一些产生自发Ca...

Watch this Scientific Journal Video about In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia Video at JoVE.com

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia Video (Video) | JoVE

该协议描述了背根神经节（DRG）的手术暴露，然后是GCaMP3（基因编码的Ca2+ 指示剂;绿色荧光蛋白-钙调蛋白-M13蛋白3）使用Pirt-GCaMP3小鼠对神经元集合进行Ca2+ 成像，同时对同侧后爪施加各种刺激。

体内 完整背根神经节中初级感觉神经元网络中神经元集合体的钙成像

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

使用这种方法监测的神经元数量允许收集神经元网络数据，这是使用旧方法或其他方法无法实现的。该技术的主要优点是能够同时监测近2， 000个初级感觉神经元，直接响应施加在后爪上的刺激。该程序特别适合研究外周异常性疼痛和疼痛超敏反应的治疗干预措施。该方法可以适用于研究三叉神经节或膝神经节，或与电压或细胞信号分子的基因编码传感器一起使用，例如环状AMP。进行这种手术需要练习。您最多可以在一只动物的六个背根神经节上练习。演示该程序将由我自己，由John Shannonhouse和Ruben Gomez先生协助。首先，将鼠标放在加热垫上，将体温保持在 37 摄氏度。通过感受小鼠的盆骨来定位腰椎增大。然后，在腰椎增大区域上方剃掉鼠标的背部。用剪刀在腰椎增大上方做一个三边矩形切口，用镊子将皮肤折叠起来。使用13毫米弹簧解剖剪刀在脊柱右侧做三到四毫米的切口。用剪刀将皮肤和肌肉剪回两侧，露出脊柱。然后，用八毫米剪刀剪掉肌肉和结缔组织，清洁右侧L5 DRG的横向过程。尽量使用棉花或凝胶泡沫吸收血液，尽量减少出血。使用弗里德曼-皮尔逊镊子或强力细钳切开右侧 L5 横向过程，注意不要接触 DRG。接下来，将鼠标和加热垫移动到自定义载物台上。使用载物台胶带将动物和加热垫固定到位。将动物的鼻子放在鼻锥中以进行连续异氟醚麻醉。将右后爪伸出到舞台外的位置固定，以便于施加刺激。将脊柱固定到位，将载物台夹在椎骨或骨盆骨的咀嚼和尾部至L5 DRG的皮肤上。调整夹具和载物台，使DRG的表面尽可能水平。然后，将载物台放在显微镜下方，使物镜在降低时直接高于DRG八毫米。插入直肠温度计。将电源线连接到加热垫和直肠温度计。将鼻锥连接到异氟醚气体管路。使用带有10x 0.4 DIC物镜的正置共聚焦显微镜和相关软件进行成像。使用绿色 FITC 滤光片设置，激发波长为 495 纳米，发射波长为 519 纳米，检测波长在 500 至 580 纳米之间。在显微镜下，找到DRG的表面。调整载物台上的夹具，使DRG表面尽可能水平，并在焦平面中可视化最大表面积。在整个过程中监测动物，以维持异氟醚麻醉而不会过量服用。要加载显微镜快速扫描协议，请使用体素大小 2.496 x 2.496 x 16 微米、512 x 512 像素、10 光学切片 Z 堆栈、一个 32 微米的 Airy 单元、1% 激光功率为 488 纳米和 5 毫瓦、像素时间 1.52 微秒、行时间 0.91 毫秒、帧时间 465 毫秒、LSM 扫描速度为 8、 双向扫描，PMT探测器增益650伏，数字增益1。通过单击“采集”选项卡下的“开始实验”，对DRG进行简短的八周期扫描。通过在一段时间内以每帧扫描一次扫描进行正交投影来创建影片。手动检查图像清晰度和成像伪影，例如穿过 DRG 的亮度波。调整夹紧位置和光学切片厚度，然后重复此步骤，直到获得清晰、高质量的电影。然后，使用体素大小 1.248 x 1.248 x 14 微米、1024 x 1024 像素、六个光学切片 Z 堆栈、1.2 艾里单位或 39 微米、5% 激光功率为 488 纳米和 25 毫瓦、像素时间 2.06 微秒、行时间 4.95 毫秒、帧时间 5.06 秒、LSM 扫描速度为 6、 双向扫描，PMT 探测器增益为 650 伏，数字增益为 1。单击“采集”选项卡下的“开始实验”按钮，制作 DRG 的高分辨率图像。加载显微镜快速扫描方案，并在DRG中记录80个周期的自发活动。生成正交投影影片，并验证图像是否具有足够的质量进行分析。要施加刺激，请将显微镜设置为执行15至20次扫描。等待扫描一到五完成以生成基线。在扫描6至10期间应用刺激。每次刺激后至少等待五分钟，然后再应用下一次刺激以防止脱敏。对于机械压力机，用爪子夹住测藻力计的夹子，不要接触爪子。在扫描 5 结束后立即开始捏住爪子，并在扫描 10 后立即停止。用测藻力计监测压力，并确保其不超过所需力的10 g。对于热刺激，将烧杯水加热到略高于所需温度。当水处于正确的温度时，将爪子浸入水中，在扫描五后立即施加刺激。扫描 10 后立即拉开烧杯。手术L5 DRG暴露，然后进行共聚焦显微镜检查，允许使用Pirt-GCaMP3小鼠一次对多达1， 800个神经元进行成像。在没有刺激的情况下，在没有刺激的情况下，在正常生理环境中对刺激做出反应的情况下，在群体水平上观察到自发钙瞬变的集合中观察到初级感觉神经元。强烈的刺激或有害的热量会增加钙的反应。与用 100 g 按压相比，用 300 g 按压增加了产生钙瞬变的神经元数量和曲线下 delta F 乘 F0 面积。在21摄氏度和45摄氏度的非有害温度范围内，暖热增加了产生钙瞬变的神经元数量和曲线下F0面积的delta F。与57摄氏度的有害热刺激相比，非有害温度激活了较少数量的神经元产生瞬态。此外，较小和中等直径的神经元在所有刺激下自发地产生钙瞬时，但大直径神经元仅在300g压力刺激下产生钙瞬变。DRG应该是水平的和最佳的光学切片厚度。它应该是这样，您可以检测到最大数量的单元格，并且电影中没有波纹。该技术已被用于在人群水平上分析躯体感觉和味觉的感觉模式，并研究成对或同步激活的相邻神经元，从而导致慢性和神经性疼痛。

Research

JoVE Journal

Neuroscience

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Functional Calcium Imaging in Developing Cortical Networks

Functional Calcium Imaging in Developing Cortical Networks (Video) | JoVE 

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

科研

神经科学

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans (Video) | JoVE 

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration (Video) | JoVE 

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals (Video) | JoVE 

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 ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb Video (Video) | JoVE

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents Video (Video) | JoVE

背根神经节的啮齿动物的脊髓和隔离的液压挤压

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration Video (Video) | JoVE

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons Video (Video) | JoVE

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release Video (Video) | JoVE

背根神经节分离与原代培养对神经递质释放的研究

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish Video (Video) | JoVE

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...