Summary

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

Published: February 10, 2023
doi:

Summary

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。

Abstract

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。

Introduction

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12。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34。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56。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78。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910,1112,1314,1516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1819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。

Protocol

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…

Representative Results

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…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

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Cite This Article
Shannonhouse, J., Gomez, R., Son, H., Zhang, Y., Kim, Y. S. In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. Vis. Exp. (192), e64826, doi:10.3791/64826 (2023).

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