Department of Cell and Molecular Physiology, UNC Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
PLoS One. 2012;7(5):e36355. doi: 10.1371/journal.pone.0036355. Epub 2012 May 1.
Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10-50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP(+) neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP(+) cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid-reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP(+) DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP(+) neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα(+) DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8(+)/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons.
降钙素基因相关肽（CGRPα，由 Calca 编码）是经典的伤害性背根神经节（DRG）神经元标志物。尽管经过多年的研究，这些神经元在体外或体内检测到什么刺激仍不清楚。为了促进这些神经元的功能研究，我们将轴突示踪剂（法呢化增强型绿色荧光蛋白；GFP）和 LoxP 停止的细胞消融构建体（人白喉毒素受体；DTR）靶向到 Calca 基因座。在培养物中，10-50%（取决于配体）的所有 CGRPα-GFP 阳性（+）神经元对辣椒素、芥末油、薄荷醇、酸性 pH 值、ATP 和瘙痒原（组胺和氯喹）有反应，表明肽能神经元在检测有害刺激和瘙痒方面发挥作用。相比之下，很少有（2.2±1.3%）CGRPα-GFP(+)神经元对 TRPM8 选择性冷却剂异丁香酚有反应。在成年小鼠中，CGRPα-GFP(+) 细胞体位于 DRG、脊髓（运动神经元和背角神经元）、大脑和甲状腺中-可重复标记所有已知表达 Calca 的细胞类型。一半的 CGRPα-GFP(+)DRG 神经元表达 TRPV1，约 25%表达神经丝-200，<10%含有非肽能标记物（IB4 和前列腺酸性磷酸酶），几乎没有（<1%）表达 TRPM8。CGRPα-GFP(+)神经元支配背侧脊髓，并支配皮肤和内脏组织。这包括表皮和保护毛上的神经末梢。我们的研究提供了直接证据，表明 CGRPα(+)DRG 神经元对引起疼痛和瘙痒的激动剂有反应，并构成了一个感觉回路，该回路与非肽能回路和 TRPM8(+) /低温回路在很大程度上不同。在未来的研究中，应该有可能条件性地消融 CGRPα 表达神经元，以评估这些神经元的感觉和非感觉功能。
