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

地球上的生命是有时间组织的，这是由于地球绕着自己的轴旋转引起的光线和温度的每日变化。因此，暴露于这些环境波动的生物体进化出每日（昼夜节律）计时器，使它们能够根据外部时间调整其行为活动和生理。生物钟是控制行为的内源性分子计时器，例如每日睡眠/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，在睡眠期间，动物更容易被捕食，睡眠也限制了觅食时间。为了最大限度地减少这些风险，系统已经到位，允许生物体在危险情况下快速醒来并执行。它们的功能是降低“唤醒阈值”，这在睡眠期间通常很高。在果蝇中，一组特殊的“唤醒神经元”已经在中央大脑中被发现。这些神经元位于大脑两侧，属于生物钟网络，由150个负责调节日常苍蝇活动的神经元组成。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其他时钟神经元指示的行为程序。在本提案中，我们希望了解唤醒神经元如何被光激活并从而唤醒动物的分子细节。由视觉系统和蓝光感光器隐花色素感知的光可以激活唤醒神经元。这种光激活会影响QUASIMODO（QSM）蛋白，该蛋白附着在唤醒神经元膜的外部。有趣的是，QSM与氯离子（Cl-）转运蛋白（NKCC）相互作用，当其活性时，导致Cl-流入细胞。在其他昆虫和哺乳动物的生物钟中，NKCC和其“拮抗剂”KCC（将Cl-转运出细胞）之间的平衡决定了GABA是作为兴奋性还是抑制性神经递质。我们推测QSM可能通过抑制黑暗中NKCC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。在光照过程中，QSM将迅速从膜上切割下来，导致NKCC抑制的释放，因此GABA现在兴奋神经元。这一假说可以解释一个可能持久的信号（GABA）如何能够迅速改变其极性以响应环境（光）的突然变化，从而降低动物的唤醒阈值。我们提出了一系列的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.