Mechanisms for light-dependent activation of circadian clock neurons in Drosophila

我们星球上的生命是暂时组织起来的，这是由于地球围绕自己的轴自转而引起的光和温度的日常变化。因此，暴露于这些环境波动的生物体进化出了每日（昼夜节律）计时器，使它们能够根据外部时间调整自己的行为活动和生理。生物钟是控制行为的内源性分子计时器，例如每天的睡眠/觉醒周期。虽然睡眠被认为对动物的表现和生存至关重要，但它也有风险。例如，动物在睡眠时更容易捕食，睡眠也限制了觅食时间。为了将这些风险降到最低，有一些系统可以让生物体迅速醒来，并在危险的情况下发挥作用。它们的功能是降低“唤醒阈值”，通常在睡眠时这个阈值很高。在果蝇中，在大脑中央发现了一组特殊的“唤醒神经元”。这些神经元，大脑两侧各有4个，属于生物钟网络，由150个神经元组成，负责调节苍蝇的日常活动。这些唤醒神经元具有明显区别于其他时钟神经元的特征，例如，它们不包含内源性昼夜节律振荡器。然而，它们与其他时钟神经元相连，并向它们传递光信息。唤醒神经元至少能够暂时覆盖由其他时钟神经元指示的行为程序。在这个提议中，我们想要了解唤醒神经元如何被光激活从而唤醒动物的分子细节。视觉系统和蓝光感受器隐花色素感知到的光可以激活唤醒神经元。这种光激活会影响附着在觉醒神经元膜外的QUASIMODO （QSM）蛋白。有趣的是，QSM与一种氯离子（Cl-）转运蛋白（NKCC）相互作用，当其活跃时，会导致Cl-流入细胞。在其他昆虫和哺乳动物的生物钟中，NKCC和它的“拮抗剂”KCC（将Cl-运输出细胞）之间的平衡决定了GABA是作为一种兴奋性神经递质还是抑制性神经递质。我们假设QSM可能通过抑制NKCC在黑暗中的功能来控制果蝇的光唤醒，从而使GABA抑制黑暗中的唤醒神经元。在光照下，QSM将迅速从膜上分离，导致NKCC抑制释放，因此GABA现在兴奋神经元。这一假设可以解释一个可能持久的信号（GABA）如何在环境（光）的突然变化下迅速改变其极性，从而降低动物的觉醒阈值。我们提出了一系列分子和行为实验来验证我们的假设。

Life on our planet is temporally organized due to the daily changes of light and temperature caused by the Earth’s rotation around its own axis. As a consequence organisms exposed to these environmental fluctuations evolved daily (circadian) timers, which allow them to adjust their behavioral activities and physiology to external time. Circadian clocks are endogenous molecular timers that control behavior, as for example the daily sleep/wake cycle. While sleep is considered as essential for the performance and survival of animals, it also bears risks. For example, during sleep animals are more prone to predation and sleep also restricts foraging time. To minimize these risks, systems are in place that allow the organism to quickly wake up and perform in dangerous situations. Their function is to lower the ‘arousal threshold’, which is normally high during sleep. In the fruit fly Drosophila, a special group of ‘arousal neurons’ has been identified in the central brain. These neurons, 4 on each side of the brain, belong to the circadian clock network, consisting of 150 neurons that are responsible for regulating daily fly activity. These arousal neurons have features that clearly distinguish them from the other clock neurons, as they for example do not contain an endogenous circadian oscillator. Nevertheless, they are connected to the other clock neurons and deliver light information to them. The arousal neurons are able to at least temporarily overwrite the behavioral program instructed by the other clock neurons.In this proposal, we want to understand the molecular details of how the arousal neurons are activated by light and thereby arouse the animal. Light perceived by the visual system and by the blue-light photoreceptor Cryptochrome can activate the arousal neurons. This light activation affects the QUASIMODO (QSM) protein, which is attached to the outside of the arousal neuron membrane. Interestingly QSM interacts with a Chloride (Cl-) transporter protein (NKCC) that, when active, leads to Cl- influx into the cell. In other insects and in the mammalian circadian clock, the balance between NKCC and its ‘antagonist’ KCC (transports Cl- out of the cell) determines if GABA acts as an exciting or inhibiting neurotransmitter. We hypothesize that QSM could control light arousal in Drosophila by inhibiting NKCC function in the dark, so that GABA inhibits the arousal neurons in darkness. During the light, QSM will be rapidly cleaved off the membrane, leading to a release of NKCC inhibition, so that GABA now excites the neuron. This hypothesis can explain how a presumably enduring signal (GABA) can quickly change its polarity in response to a sudden change in the environment (light), and thereby lower the arousal threshold of the animal. We propose an array of molecular and behavioral experiments to test our hypothesis.