The activity and the metabolism of the brain change rhythmically during the day/night cycle. Such rhythmicity is also observed in cultured neurons from the suprachiasmatic nucleus, which is a critical center in rhythm maintenance. However, this issue has not been extensively studied in cultures from areas less involved in timekeeping, as the hippocampus. Using neurons cultured from the hippocampi of newborn rats (both male and female), we observed significant time-dependent changes in global activity, in synaptic vesicle dynamics, in synapse size, and in synaptic mRNA amounts. A transcriptome analysis of the neurons, performed at different times over 24 h, revealed significant changes only for RNA-binding motif 3 (Rbm3). RBM3 amounts changed, especially in synapses. RBM3 knockdown altered synaptic vesicle dynamics and changed the neuronal activity patterns. This procedure also altered local translation in synapses, albeit it left the global cellular translation unaffected. We conclude that hippocampal cultured neurons can exhibit strong changes in their activity levels over 24 h, in an RBM3-dependent fashion.

SIGNIFICANCE STATEMENT This work is important in several ways. First, the discovery of relatively regular activity patterns in hippocampal cultures implies that future studies using this common model will need to take the time parameter into account, to avoid misinterpretation. Second, our work links these changes in activity strongly to RBM3, in a fashion that is independent of the canonical clock mechanisms, which is a very surprising observation. Third, we describe here probably the first molecule (RBM3) whose manipulation affects translation specifically in synapses, and not at the whole-cell level. This is a key finding for the rapidly growing field of local synaptic translation.

在白天/夜晚周期中，大脑的活动和新陈代谢有节奏地变化。在视交叉上核的培养神经元中也观察到这种节律性，这是节律维持的关键中心。但是，在海马等较少涉及计时的地区的文化中，尚未对该问题进行广泛研究。使用从新生大鼠（雄性和雌性）海马体培养的神经元，我们观察到了全球活动，突触囊泡动力学，突触大小和突触mRNA量的显着时间依赖性变化。在24小时内的不同时间进行的神经元转录组分析显示，仅RNA结合基序3（Rbm3）发生了显着变化。RBM3的数量发生了变化，尤其是在突触中。RBM3组合式改变突触小泡动力学，并改变神经元活动模式。此过程也改变了突触中的局部翻译，尽管它不影响整体细胞翻译。我们得出的结论是，海马培养的神经元可以在24小时内以依赖RBM3的方式在其活动水平上表现出强烈的变化。

意义声明这项工作在几个方面都很重要。首先，在海马文化中发现相对规律的活动模式意味着使用该通用模型进行的未来研究将需要考虑时间参数，以避免产生误解。其次，我们的工作将活动的这些变化与RBM3紧密联系在一起，其方式与规范时钟机制无关，这是非常令人惊讶的发现。第三，我们可能在这里描述了第一个分子（RBM3），其操作会特别在突触中而不是在全细胞水平上影响翻译。这是快速增长的本地突触翻译领域的关键发现。