Department of Oral & Maxillofacial Surgery, School of Dentistry, University of Texas Health Science Center at San Antonio.
Department of Oral & Maxillofacial Surgery, School of Dentistry, University of Texas Health Science Center at San Antonio; Programs in Integrated Biomedical Sciences, Translational Sciences, Biomedical Engineering, Radiological Sciences, University of Texas Health Science Center at San Antonio;
J Vis Exp. 2023 Feb 10(192). doi: 10.3791/64826.
Ca imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca entry into the cytoplasm or the release of intracellular Ca stores. Pirt-GCaMP3-based Ca imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca transients as well as the amplitude of Ca transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, Aβ, Aδ, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.
钙成像可以作为细胞活动的替代指标，包括动作电位和涉及细胞质中 Ca 进入或细胞内 Ca 库释放的各种信号机制。基于 Pirt-GCaMP3 的小鼠背根神经节 (DRG) 感觉神经元钙成像具有同时测量大量细胞的优势。最多可以监测 1800 个神经元，允许在正常生理环境下以群体水平在体内研究神经元网络和躯体感觉过程。监测的大量神经元可以检测到其他方法难以检测到的活动模式。可以向小鼠后爪施加刺激，从而可以研究刺激对 DRG 神经元群体的直接影响。产生钙瞬变的神经元数量以及钙瞬变的幅度表明对特定感觉模式的敏感性。神经元的直径提供了激活纤维类型的证据（非伤害性机械 vs. 伤害性疼痛纤维、Aβ、Aδ 和 C 纤维）。可以用 td-Tomato 和特定的 Cre 重组酶与 Pirt-GCaMP 一起对表达特定受体的神经元进行基因标记。因此，DRG 的 Pirt-GCaMP3 钙成像为分析特定感觉模式和作为群体水平上的神经元亚型提供了一种强大的工具和模型，用于研究疼痛、瘙痒、触觉和其他躯体感觉信号。
