Do single neurons need to sleep and why? Investigating the cellular signatures o

摘要 睡眠被认为是恢复大脑功能的关键，而越来越多的证据表明，睡眠的一个关键 功能可能是在觉醒过程中重新平衡由可塑性引发的细胞变化。这一证据是一致的 假设睡眠和醒来可以发生，被调节，并在水平上执行其功能 单个神经元。最近，利用自由活动大鼠的多阵列记录，我们获得了直接的证据 睡眠可以发生在一组皮质神经元的局部，而大脑的其余部分保持清醒，并且 这种“局部睡眠”会随着清醒时间的延长而增加。如果是这样的话，就会出现很多问题：局部睡眠也会吗？ 发生在与哺乳动物非常不同的物种中，比如苍蝇？是发生的机制吗？ 局部睡眠类似于已知的调节睡眠需求的睡眠，特别是强烈的神经可塑性导致 疲倦，这需要睡眠来强制突触重整化？或者局部睡眠反映的是暂时的 由于强烈的神经活动和能量或钙储存的短期耗尽而导致的神经元疲劳，因此 不符合睡眠的标准？最后，尚不清楚是否有细胞/超微结构特征 睡眠已经发生，并可能发挥了它的功能。在这项提案中，我们将测试局部睡眠 通过使用活体钙成像来同时监测苍蝇体内数十个神经元的活动 特定的神经元回路。然后，我们将测试局部睡眠是否像正常睡眠一样，受到高强度睡眠的调节 突触可塑性(“疲劳”)或仅仅通过活动(“疲劳”)。要做到这一点，我们首先要确定本地 在复杂的丰富环境(Fly Mall)中，延长清醒时间会增加睡眠，预计会导致 蘑菇体的疲劳，相对于在贫穷环境(单管)中延长的尾流。 接下来，我们将在单管延长尾流期间以及在 睡吧。这两种剧烈活动而塑性很小的情况预计会导致疲劳，以及它们的影响 对局部睡眠的影响将再次与单管延长尾流的情况进行比较。为了进一步使 可塑性的影响从那些活性我们将重复的实验中学习突变体，在其中暴露 去逛商场不应该导致可塑性/疲劳感。最后，我们将使用SBF-SEM来测试是否有 能够区分醒着的果蝇和清醒果蝇的神经元的超微结构特征 睡眠，并比较具有可塑性的清醒导致疲劳的效果与清醒与可塑性相关的 DTrpA1介导的强烈刺激导致疲劳。总之，这些对苍蝇的研究将是对 项目II中的小鼠，使用类似或非常相同的方法。在一起，他们将确定是否睡觉和醒来 在单个神经元水平上进行自我平衡调节，如果它们留下了反映 它们对单个细胞的影响和作用。

ABSTRACT Sleep is thought to be essential to restore brain functions, and converging evidence suggests that a key function may be to rebalance cellular changes triggered by plasticity during wake. This evidence is consistent with the hypothesis that sleep and wake may occur, be regulated, and perform their functions at the level of individual neurons. Recently, using multi-array recordings in freely moving rats, we obtained direct evidence that sleep can occur locally within a group of cortical neurons, while the rest of the brain remains awake, and that such "local sleep" increases with the duration of wake. If so, many questions arise: does local sleep also occur in species very different from mammals, such as flies? Are the mechanisms underlying the occurrence of local sleep similar to those that are known to regulate sleep need, specifically intense neural plasticity leading to tiredness, which requires sleep to enforce synaptic renormalization? Or does local sleep reflect temporary neuronal fatigue due to intense neural activity and short-lasting depletion of energy or calcium stores, and thus not qualify properly as sleep? Finally, it is unknown whether there are cellular/ultrastructural signatures that sleep has occurred and presumably performed its functions. In this proposal, we will test whether local sleep exists in flies by using in vivo calcium imaging to monitor simultaneously the activity of dozens of neurons in specific neuronal circuits. We will then test whether local sleep, like sleep proper, is regulated by intense synaptic plasticity ("tiredness") or instead by mere activity ("fatigue"). To do so we will first establish if local sleep increases during extended wake in a complex enriched environment (fly mall), expected to lead to tiredness in the mushroom bodies, relative to extended wake in an impoverished environment (single tube). Next, we will induce local dTrpA1-mediated activation during extended wake in single tubes, as well as during sleep. These 2 conditions of intense activity with little plasticity are expected to lead to fatigue, and their effects on local sleep will again be compared with those of extended wake in single tubes. To further decouple the effects of plasticity from those of activity we will repeat the experiments in learning mutants, in which exposure to the fly mall should not lead to plasticity/tiredness. Finally, we will use SBF-SEM to test whether there are ultrastructural signatures that can distinguish neurons of flies that have been awake from those of flies that slept, and compare the effects of wake with plasticity leading to tiredness with those of wake associated with dTrpA1-mediated intense firing leading to fatigue. Altogether, these studies in flies will complement those in mice in Project II, which use similar or the very same methods. Together, they will establish if sleep and wake are regulated homeostatically at the single neuron level, and if they leave ultrastructural signatures that reflect their consequences and functions for individual cells.