A sleep- and locomotion stop neuron with compartmentalized Ca2+ dynamics as a CPG regulator?

动物需要能够主动地结束运动，以便等待特定的事件，或者重新启动不同方向的运动，例如在导航期间。当动物进入睡眠状态时，它们也需要停止运动，这通常是通过不同的机制发生的。然而，线虫线虫的一个神经元，RIS，参与了幼虫阶段的发育定时睡眠，并且，正如我们可以显示的那样，在成年动物中也扮演着运动停止神经元的角色。RIS活动恰好发生在减速之前，并进一步参与指示逆转。RIS显示了沿着轴突的分隔的钙活动：当终末部分在减慢发生时变得活跃，而轴突的腹侧支只有在减慢事件之后发生反转时才显示活动。通过光遗传刺激，我们发现RIS通过释放GABA和FLP-11神经肽而停止运动。后者参与抑制运动神经元的有节奏/同步活动，作为运动模式生成器的一部分。然而，这一方面还没有得到详细的澄清。此外，我们还不知道RIS与线虫神经系统中其他神经元的确切相互作用，这些神经元共同启动和调节运动和逆转。在我们先前建议的这个扩展中，我们想要与其他神经元一起讨论RIS的功能。初步数据显示，在移动的动物中，RIS和RIM神经元(后者参与协调逆转行为)中的钙活动同时成像，显示了这些细胞的活跃相互作用，令人惊讶的是，RIS的活动先于RIM。此外，通过电压成像，我们显示了RIS与RIM和AVJ神经元的活跃的缝隙连接，这两个神经元在解剖上与RIS轴突的分支有联系。我们将详细分析这些交互作用。我们还将讨论RIM与PVC前向命令中间神经元的相互作用，这些神经元在化学上突触到RIS分支，并可能抑制RIS以防止逆转，如此类事件之前活动减少所示。利用电压成像，我们发现RIM和RIS神经元表现出紧密耦合，但倒易膜电位变化，可能是通过整顿缝隙连接介导的。我们将分析这些相互作用及其对不同分子角色(缝隙连接亚单位、神经肽受体)的依赖。此外，我们还将评估RIS光刺激后腹神经索上运动神经元的电活动。我们预计这将以不同的方式影响不同的运动神经元组，使我们能够发现模式生成的特定影响。分析RIS在睡眠和运动停止中的功能，以及它是如何解耦模式生成器的，将有助于理解这些基本功能是如何进化并分布到高等动物的不同大脑系统的。这些基本功能存在于紧凑的线虫神经系统的一个神经元中。

Animals need to be able to actively end locomotion, in order to await certain events, or to re-initiate locomotion in a different direction, e.g. during navigation. Animals also need to stop locomotion when they enter sleep, and typically this occurs by different mechanisms. However, a single neuron of the nematode Caenorhabditis elegans, RIS, is involved in orchestrating developmentally timed sleep in larval stages, and, as we could show, also acts as a locomotion stop neuron in adult animals. RIS activity occurs right before slowing, and is further involved in instructing reversals. RIS shows compartmentalized Ca2+ activity along its axon: While the terminal part becomes active whenever slowing occurs, a ventral branch of the axon shows activity only when the slowing event is followed by a reversal. By optogenetic stimulation we showed that RIS stops locomotion by releasing GABA and FLP-11 neuropeptides. The latter is involved in silencing rhythmic / synchronized activity of motor neurons, as part of pattern generators for locomotion. However, this aspect is not clarified in detail yet. Furthermore, we do not know the exact interplay of RIS with other neurons in the C. elegans nervous system, that cooperate to initiate and regulate locomotion and reversals. In this extension of our previous proposal, we want to address the function of RIS in concert with other neurons. Preliminary data, where we concomitantly imaged Ca2+ activity in RIS and RIM neurons (the latter are involved in orchestrating reversal behavior) in moving animals, shows an active interplay of these cells, where RIS activity, surprisingly, precedes RIM. Furthermore, by voltage imaging, we show active gap junction connections of RIS to RIM and AVJ neurons, which have anatomical connections to the branch of the RIS axon. We will analyze these interactions in detail. We will also address interactions of RIM with PVC forward command interneurons, that chemically synapse onto the RIS branch, and which may inhibit RIS in order to prevent reversals, as indicated by reduced activity preceding such events. Using voltage imaging, we found that RIM and RIS neurons show tightly coupled, but reciprocal membrane potential changes, possibly mediated by rectifying gap junctions. We will analyze these interactions and their dependence on distinct molecular players (gap junction subunits, neuropeptide receptors). Also, we will assess electrical events in motor neurons along the ventral nerve cord, following RIS photostimulation. We expect this to influence different groups of motor neurons differently, enabling us to uncover specific effects in pattern generation.Analyzing the functions of RIS in sleep and locomotion stop, and how it uncouples pattern generators, will help understanding how these fundamental functions, present in one neuron in the compact C. elegans nervous system, may have evolved and distributed to distinct brain systems in higher animals.