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

动物需要能够主动结束运动，以便等待某些事件，或者在不同的方向重新开始运动，例如在导航过程中。动物在进入睡眠时也需要停止运动，这通常是通过不同的机制发生的。然而，秀丽隐杆线虫（cenorhabditis elegans， RIS）的一个神经元参与了幼虫阶段协调发育定时睡眠的过程，而且，正如我们所展示的，在成年动物中也起着运动停止神经元的作用。RIS活动发生在减速之前，并进一步参与指示逆转。RIS沿其轴突显示出区隔化的Ca2+活动：当减慢发生时，终端部分变得活跃，轴突的腹侧分支仅在减慢事件发生后发生逆转时才显示出活动。通过光遗传刺激，我们发现RIS通过释放GABA和FLP-11神经肽来停止运动。后者参与抑制运动神经元的节律/同步活动，作为运动模式发生器的一部分。然而，这方面还没有得到详细的阐明。此外，我们不知道RIS与秀丽隐杆线虫神经系统中其他神经元的确切相互作用，这些神经元合作启动和调节运动和逆转。在我们之前的提议的扩展中，我们想要解决RIS与其他神经元协同的功能。我们在运动动物的RIS和RIM神经元（后者参与协调逆转行为）中同时成像的初步数据显示，这些细胞之间存在活跃的相互作用，令人惊讶的是，RIS活性先于RIM。此外，通过电压成像，我们发现RIS与RIM和AVJ神经元之间存在活跃的间隙连接，而RIM和AVJ神经元与RIS轴突分支具有解剖学上的联系。我们将详细分析这些相互作用。我们还将讨论RIM与PVC前向指令中间神经元的相互作用，这些神经元化学突触连接到RIS分支上，并可能抑制RIS以防止逆转，正如在此类事件发生之前活性降低所表明的那样。通过电压成像，我们发现RIM和RIS神经元表现出紧密耦合但相互的膜电位变化，可能是由整流间隙连接介导的。我们将分析这些相互作用及其对不同分子参与者（间隙连接亚基，神经肽受体）的依赖。此外，我们将评估沿腹侧神经索运动神经元在RIS光刺激后的电事件。我们期望这会对不同组的运动神经元产生不同的影响，使我们能够发现模式生成中的特定影响。分析RIS在睡眠和运动停止中的功能，以及它如何解耦模式生成器，将有助于理解这些存在于紧凑的秀丽隐杆线虫神经系统中的一个神经元中的基本功能是如何进化并分布到高等动物的不同大脑系统中的。

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.