State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Microsc Res Tech. 2024 Dec;87(12):2894-2914. doi: 10.1002/jemt.24662. Epub 2024 Jul 23.
Serotonin (5-hydroxytryptamine, 5-HT) is a very important neurotransmitter emerging from the raphe nuclei to several brain regions. Serotonergic neuronal connectivity has multiple functions in the brain. In this study, several techniques were used to trace serotonergic neurons in the dorsal raphe (DR) and median raphe (MnR) that project toward the arcuate nucleus of the hypothalamus (Arc), dorsomedial hypothalamic nucleus (DM), lateral hypothalamic area (LH), paraventricular hypothalamic nucleus (PVH), ventromedial hypothalamic nucleus (VMH), fasciola cinereum (FC), and medial habenular nucleus (MHb). Cholera toxin subunit B (CTB), retro-adeno-associated virus (rAAV-CMV-mCherry), glycoprotein-deleted rabies virus (RV-ΔG), and simultaneous microinjection of rAAV2-retro-Cre-tagBFP with AAV-dio-mCherry in C57BL/6 mice were used in this study. In addition, rAAV2-retro-Cre-tagBFP was microinjected into Ai9 mice. Serotonin immunohistochemistry was used for the detection of retrogradely traced serotonergic neurons in the raphe nuclei. The results indicated that rAAV2-retro-Cre-tagBFP microinjection in Ai9 mice was the best method for tracing serotonergic neuron circuits. All of the previously listed nuclei exhibited serotonergic neuronal projections from the DR and MnR, with the exception of the FC, which had very few projections from the DR. The serotonergic neuronal projections were directed toward the Arc by the subpeduncular tegmental (SPTg) nuclei. Moreover, the RV-ΔG tracer revealed monosynaptic non-serotonergic neuronal projections from the DR that were directed toward the Arc. Furthermore, rAAV tracers revealed monosynaptic serotonergic neuronal connections from the raphe nuclei toward Arc. These findings validate the variations in neurotropism among several retrograde tracers. The continued discovery of several novel serotonergic neural circuits is crucial for the future discovery of the functions of these circuits. RESEARCH HIGHLIGHTS: Various kinds of retrograde tracers were microinjected into C57BL/6 and Ai9 mice. The optimum method for characterizing serotonergic neuronal circuits is rAAV2-retro-Cre-tagBFP microinjection in Ai9 mice. The DR, MnR, and SPTg nuclei send monosynaptic serotonergic neuronal projections toward the arcuate nucleus of the hypothalamus. Whole-brain quantification analysis of retrograde-labeled neurons in different brain nuclei following rAAV2-retro-Cre-tagBFP microinjection in the Arc, DM, LH, and VMH is shown. Differential quantitative analysis of median and dorsal raphe serotonergic neurons emerging toward the PVH, DM, LH, Arc, VMH, MHb, and FC is shown.
血清素（5-羟色胺，5-HT）是一种非常重要的神经递质，它从中缝核投射到大脑的几个区域。 5-羟色胺能神经元的连接在大脑中具有多种功能。 在这项研究中，使用了几种技术来追踪投射到下丘脑弓状核（Arc）、中缝核（MnR）、背内侧下丘脑核（DM）、外侧下丘脑区（LH）、室旁下丘脑核（PVH）、腹内侧下丘脑核（VMH）、灰结节（FC）和内侧缰状核（MHb）的背侧中缝核（DR）和中缝核（MnR）中的 5-羟色胺能神经元。 在 C57BL/6 小鼠中使用了霍乱毒素亚单位 B（CTB）、逆行腺相关病毒（rAAV-CMV-mCherry）、糖蛋白缺失狂犬病病毒（RV-ΔG）和同时微注射 rAAV2- retro-Cre-tagBFP 与 AAV-dio-mCherry 在 Ai9 小鼠中。 此外，rAAV2-retro-Cre-tagBFP 被微注射到 Ai9 小鼠中。 血清素免疫组织化学用于检测中缝核中逆行追踪的 5-羟色胺能神经元。 结果表明，rAAV2-retro-Cre-tagBFP 微注射到 Ai9 小鼠中是追踪 5-羟色胺能神经元回路的最佳方法。 除了 FC 之外，所有之前列出的核都显示出来自 DR 和 MnR 的 5-羟色胺能神经元投射，而 FC 则很少有来自 DR 的投射。 5-羟色胺能神经元投射通过脚间核被盖（SPTg）核指向弓状核。 此外，RV-ΔG 示踪剂显示来自 DR 的单突触非 5-羟色胺能神经元投射，其方向指向 Arc。 此外，rAAV 示踪剂显示来自中缝核的单突触 5-羟色胺能神经元连接到 Arc。 这些发现验证了几种逆行示踪剂之间神经亲嗜性的变化。 未来发现这些回路的功能，需要继续发现几种新的 5-羟色胺能神经回路。 研究亮点： 将各种逆行示踪剂微注射到 C57BL/6 和 Ai9 小鼠中。 表征 5-羟色胺能神经元回路的最佳方法是在 Ai9 小鼠中微注射 rAAV2-retro-Cre-tagBFP。 DR、MnR 和 SPTg 核发出单突触 5-羟色胺能神经元投射到下丘脑弓状核。 显示 rAAV2-retro-Cre-tagBFP 微注射到 Arc、DM、LH 和 VMH 后，不同脑核中逆行标记神经元的全脑定量分析。 显示从中缝核出现的中缝和背侧 5-羟色胺能神经元向 PVH、DM、LH、Arc、VMH、MHb 和 FC 的差异定量分析。
