Li Xuan, Yao Juan, Zeng Lian-Lin, Huang Jia, Jiang Xiao-Qian, Xu Jin-He, Wu Bing, Li Hong-Li, Hu Ke-Hui
Experimental Center of Basic Medicine, Army Medical University, Chongqing 400038, China.
Department of rehabilitation Medicine, Suining Central Hospital, Suining 629000, China.
Brain Res Bull. 2025 Sep;229:111474. doi: 10.1016/j.brainresbull.2025.111474. Epub 2025 Jul 22.
Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool for controlling targeted cell populations and manipulating brain functions. As a typical chemogenetic actuator in DREADD systems, clozapine-N-oxide (CNO) is widely utilized in neurobiological and pharmacological research. However, the acute off-target effects of CNO on somatosensory (e.g., itch, pain) stimulation-induced and anxiety-related behaviors in rodents have not been systematically investigated. Through classical behavioral paradigms, including evoked itch-scratching, noxious thermal pain assessment, and elevated plus maze testing, we demonstrated that intraperitoneal administration of standard CNO doses (2 and 4 mg/kg) modulates itch/pain perception and anxiety-like behaviors in Sprague-Dawley rats lacking DREADD expression. The key behavioral findings are: (1) High-dose CNO (4 mg/kg) significantly suppressed 5-HT-induced itch-scratching; (2) Both doses of CNO (2 and 4 mg/kg) reduced sensitivity to noxious thermal stimuli; (3) CNO induced an anxious phenotype without altering locomotor activity. Fiber photometry experiments further revealed that CNO enhanced glutamatergic neuronal activity in the prelimbic cortex but not in the central amygdala or the dorsal hippocampus. These results provide robust evidence that commonly employed doses of CNO-designed to activate DREADDs-elicit off-target effects on somatosensory and emotional behaviors in rats. The underlying mechanisms may involve CNO-induced modulation of glutamatergic signaling in key limbic regions, potentially mediated by interactions between the ligand or its metabolites and endogenous brain receptors. Verification of these off-target effects underscores the necessity of including ligand-only control groups in DREADD experiments and necessitates caution against overinterpreting behavioral outcomes attributed solely to chemogenetic manipulations.
仅由设计药物激活的设计受体（DREADDs）是一种用于控制靶向细胞群和操纵脑功能的强大化学遗传工具。作为DREADD系统中的典型化学遗传激活剂，氯氮平 - N - 氧化物（CNO）被广泛应用于神经生物学和药理学研究。然而，CNO对啮齿动物体感（如瘙痒、疼痛）刺激诱导的行为和焦虑相关行为的急性脱靶效应尚未得到系统研究。通过经典行为范式，包括诱发瘙痒搔抓、有害热痛评估和高架十字迷宫测试，我们证明腹腔注射标准剂量的CNO（2和4mg/kg）可调节缺乏DREADD表达的Sprague - Dawley大鼠的瘙痒/疼痛感知和焦虑样行为。关键行为学发现如下：（1）高剂量CNO（4mg/kg）显著抑制5 - HT诱导的瘙痒搔抓；（2）两种剂量的CNO（2和4mg/kg）均降低了对有害热刺激的敏感性；（3）CNO诱导焦虑表型但不改变运动活性。光纤光度法实验进一步表明，CNO增强了前边缘皮层中的谷氨酸能神经元活性，但在中央杏仁核或背侧海马体中未增强。这些结果提供了有力证据，表明常用于激活DREADDs的CNO剂量会对大鼠的体感和情绪行为产生脱靶效应。潜在机制可能涉及CNO诱导的关键边缘区域谷氨酸能信号调节，可能由配体或其代谢物与内源性脑受体之间的相互作用介导。对这些脱靶效应的验证强调了在DREADD实验中纳入仅使用配体的对照组的必要性，并提醒人们谨慎对待仅归因于化学遗传操作的行为结果的过度解读。
