Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide synthesized by posterior hypothalamic and incerto-hypothalamic neurons that project throughout the central nervous system. The MCHergic system modulates several important functions such as feeding behavior, mood and sleep. MCH exerts its biological functions through interaction with the MCHR-1 receptor, the only functional receptor present in rodents. The internalization process of MCHR-1 triggered by MCH binding was described in vitro in non-neuronal heterologous systems with over-expression of MCHR-1. Reports of in vivo MCHR-1 internalization dynamics are scarce, however, this is an important process to explore based on the critical functions of the MCHergic system. We had previously determined that 60 min after intracerebroventricular (i.c.v.) microinjections of MCH conjugated with fluorophore rhodamine (R-MCH), the dorsal and median raphe nucleus presented R-MCH positive labeled neurons. In the present work, we further studied the in vivo uptake process focusing on the distribution and time-dependent pattern of R-MCH positive cells 10, 20 and 60 min (T10, T20 and T60, respectively) after i.c.v. microinjection of R-MCH. We also explored this uptake process to see whether it was receptor- and clathrin-dependent and examined the phenotype of R-MCH positive cells and their proximity to MCHergic fibers. We found a great number of R-MCH positive cells with high fluorescence intensity in the lateral septum, nucleus accumbens and hippocampus at T20 and T60 (but not at T10), while a lower number with low intensity was observed in the dorsal raphe nucleus. At T20, in rats pre-treated with a MCHR-1 antagonist (ATC-0175) or with phenylarsine oxide (PAO), a clathrin endocytosis inhibitor, a robust decrease (> 50 %) of R-MCH uptake occurred in these structures. The R-MCH positive cells were identified as neurons (NeuN positive, GFAP negative) and some MCHergic fibers run in the vicinities of them. We concluded that neurons localized at structures that were close to the ventricular surfaces could uptake R-MCH in vivo through a receptor-dependent and clathrin-mediated process. Our results support volume transmission of MCH through the cerebrospinal fluid to reach distant targets. Finally, we propose that R-MCH would be an effective tool to study MCH-uptake in vivo.

黑色素浓缩激素 (MCH) 是一种下丘脑神经肽，由投射到整个中枢神经系统的下丘脑后部和下丘脑内神经元合成。MCHergic 系统调节几个重要的功能，例如进食行为、情绪和睡眠。MCH 通过与啮齿动物中唯一的功能性受体 MCHR-1 受体相互作用发挥其生物学功能。在 MCHR-1过表达的非神经元异源系统中体外描述了由 MCH 结合触发的 MCHR-1 内化过程。体内报告MCHR-1 内化动力学是稀缺的，然而，这是基于 MCHergic 系统的关键功能探索的重要过程。我们之前已经确定，在脑室内 (icv) 显微注射与荧光团罗丹明 (R-MCH) 结合的 MCH 后 60 分钟，背侧和中缝核呈现 R-MCH 阳性标记的神经元。在目前的工作中，我们进一步研究了体内在 icv 微量注射 R-MCH 后 10、20 和 60 分钟（分别为 T10、T20 和 T60）的 R-MCH 阳性细胞的分布和时间依赖性模式的摄取过程。我们还探索了这种摄取过程，看看它是否依赖于受体和网格蛋白，并检查了 R-MCH 阳性细胞的表型及其与 MCHergic 纤维的接近程度。我们在T20和T60（但不是在T10）在侧隔、伏核和海马中发现了大量具有高荧光强度的R-MCH阳性细胞，而在中缝背核中观察到数量较少且强度较低的R-MCH阳性细胞。在 T20 时，在用 MCHR-1 拮抗剂 (ATC-0175) 或氧化苯胂 (PAO)（一种网格蛋白内吞作用抑制剂）预处理的大鼠中，这些结构中的 R-MCH 摄取显着下降（> 50%）。R-MCH阳性细胞被鉴定为神经元（NeuN阳性，GFAP阴性），一些MCHergic纤维在它们附近运行。我们得出结论，位于靠近心室表面的结构的神经元可以摄取 R-MCH在体内通过受体依赖性和网格蛋白介导的过程。我们的结果支持 MCH 通过脑脊液的体积传输到达远处目标。最后，我们建议 R-MCH 将成为研究体内 MCH 摄取的有效工具。