| 张 坤,王建刚,王静怡,罗 静,范 蕾,程傲冰,韩燕飞.两种基因编码的探针特异性检测小鼠脑5-HT信号的比较[J].,2022,(14):2601-2606 |
| 两种基因编码的探针特异性检测小鼠脑5-HT信号的比较 |
| Comparison of Two Genetically Encoded Fluorescent Sensors to Detect 5-HT in Mouse Brain |
| 投稿时间：2021-12-23 修订日期：2022-01-17 |
| DOI：10.13241/j.cnki.pmb.2022.14.001 |
| 中文关键词: 基因编码探针 GPCR PBPs 5-HT |
| 英文关键词: Genetically encoded sensor GPCR PBPs 5-HT |
| 基金项目:国家自然科学基金项目（82101284 81701070）；河南省科技攻关项目（212102310824） |
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| 中文摘要: |
| 摘要 目的：五羟色胺（5-HT）对中枢神经系统发挥着重要的调控作用，其功能失调与许多精神类疾病密切相关，因此开发能实时灵敏检测5-HT的工具对研究此类疾病及研发靶向药物至关重要。最近新开发的iSeroSnFR和GRAB5-HT1.0两类5-HT荧光探针，具有细胞特异性、反应灵敏以及高时空分辨率等显著优势，已经成为深入探究5-HT作用机制的有力工具。本研究旨在探究荧光探针iSeroSnFR和GRAB5-HT1.0哪种能更有效地检测5-HT递质释放及动态变化，从而为后续优化5-HT荧光探针和利用这一有力工具解析神经疾病中的递质异常提供新思路。方法：将iSeroSnFR或GRAB5-HT1.0探针的基因序列插入到Sindbis病毒表达载体，然后把病毒分别转染到培养的小鼠离体脑片的海马CA1区，比较两种探针在神经元中的表达差异；并检测其对外源性5-HT药物诱导产生的荧光反应。另外，将上述病毒转染到急性小鼠脑片中缝核（DRN）区，给与电刺激检测两种探针响应内源5-HT释放的荧光信号差异。结果：在转染iSeroSnFR的海马CA1区神经元中均有荧光表达，但细胞边界不清晰；而转染了GRAB5-HT1.0的神经元细胞膜上表达有强烈的绿色荧光信号。用1 μM 5-HT处理培养的小鼠脑片海马CA1神经元时，iSeroSnFR探针没有明显的荧光强度变化；而GRAB5-HT1.0探针产生的荧光强度显著增强；用1 mM 5-HT处理时，iSeroSnFR探针产生一定强度的荧光反应，但响应幅度弱于GRAB5-HT1.0探针。另外，利用上述病毒转染急性小鼠脑片DRN区，通过电刺激诱导内源性5-HT释放，发现仅在表达GRAB5-HT1.0探针的DRN神经元中观察到显著增强的荧光反应；而iSeroSnFR探针几乎未产生明显荧光变化。结论：在小鼠脑中，GRAB5-HT1.0荧光探针对外源性及内源性的5-HT的亲和力更高、灵敏度更强。 |
| 英文摘要: |
| ABSTRACT Objective: 5-HT (5-hydroxytryptamine) is a monoamine neurotransmitter in the brain, playing a critical role in a broad range of biological processes. 5-HT dysregulation has been involved in mental disorders, including anxiety and depression. Despite the importance of 5-HT, our understanding of cell-specific 5-HT signaling is flawed, partly due to the inability to measure 5-HT with a high spatiotemporal solution. Recently developed iSeroSnFR and GRAB5-HT1.0, two genetically encoded 5-HT sensors, have attractive features because of their cell specificity, sensitivity, and excellent spatiotemporal resolution to probe 5-HT dynamics. Our study aims to compare the different properties of iSeroSnFR and GRAB5-HT1.0 in response to 5-HT in the mouse brain. It can provide new insights into the optimization of 5-HT sensors and the development of other genetically encoded fluorescent neurotransmitter sensors. It can also help us choose the appropriate sensor based on different scientific issues and explore molecular and cellular mechanisms of neurological diseases. Methods: The iSeroSnFR sensor or GRAB5-HT1.0 sensor was expressed in the CA1 neurons of cultured hippocampal slices or DRN of acute mouse brain slices using the Sindbis viral expression system. Then the fluorescence intensity and the fluorescent changes of iSeroSnFR and GRAB5-HT1.0 in response to 5-HT drug puff on cultured mouse hippocampal slices or electrical stimuli evoked 5-HT release on acute mouse DRN slices were compared. Results: We found many fluorescence signals in iSeroSnFR-expressing hippocampal CA1 neurons, but the cell boundary was unclear. In comparison, there was a robust green fluorescence signal in GRAB5-HT1.0-expressing hippocampal CA1 neurons. Subsequently, when the CA1 region of mouse hippocampal cultured slices was exposed to 1 μM 5-HT application, the iSeroSnFR sensor almost had no detectable fluorescence changes. However, GRAB5-HT1.0 sensor produced robust fluorescence responses. Meanwhile the fluorescence response of the iSeroSnFR is weaker than that of the GRAB5-HT1.0 in response to puffed 1 mM 5-HT. In addition, we made Sindbis viral expression of iSeroSnFR or GRAB5-HT1.0 sensor and applied electrical stimuli to evoke 5-HT release in the DRN region of acute mouse brain slices. It provoked robust increases in fluorescence in DRN slices expressing GRAB5-HT1.0 sensor. On the contrary, the same electrical stimuli almost have no effect in DRN slices expressing the iSeroSnFR sensor. Conclusion: The GRAB5-HT1.0 sensor can measure the exogenous 5-HT drug and endogenous 5-HT release with higher affinity and sensitivity in the mouse brain. |
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