How do neural microcircuits and networks in the suprachiasmatic nucleus encode circadian time?

共同的经验告诉我们，我们是24小时的生命。最明显的是，我们每天都在睡觉和醒来，而在白天和黑夜的过程中，我们的情绪状态、认知能力和总体能量水平在定期的节目中起伏不定。这些节奏与日出和日落的时间有关，但当我们在不同时区之间旅行或轮换班次时，我们会意识到它们也依赖于内在的暗示。事实上，进化为我们和几乎所有类型的生物提供了内部生物钟，这些生物钟控制着我们的日常周期，并受到阳光的提示。哺乳动物的主要内钟是大脑下丘脑的视交叉上核(SCN)。它从眼睛接受神经输入，以提示它进入黑暗和光明的周期，但它不需要这种输入来产生自己的神经元活动周期，周期大约为(大约)一天：因此它是一个“昼夜节律”时钟。在过去的20年里，昼夜节律生物学的主要进展之一是识别了构成昼夜节律计时器的基因和蛋白质。在SCN的每个细胞中，这些基因和它们编码的蛋白质被锁定在一个自我维持的蛋白质合成反馈环路中，随后蛋白质降解。这些过程的生物化学意味着这个循环大约一天完成一次。这是一种细胞昼夜节律机制，驱动SCN中平行的神经元活动昼夜周期，这些神经元活动周期反过来将日常时间传递给大脑和身体的其余部分。尽管在解开细胞发条的“螺母和螺栓”方面取得了这一显著的突破，但关于SCN的许多基本问题仍然没有得到回答。例如，SCN由20,000个细胞组成，每个细胞的发条与其他细胞的发条紧密相连。如果SCN要给身体一个明确的时间信号，这是一个必要的属性--但它是如何实现的呢？其次，尽管它们是同步的，但各个细胞的时钟并不是同时活跃的。当培养皿中培养成“器官型”切片的SCN中的神经元活动在显微镜下成像时，可以看到每天都有一波又一波的活动席卷组织。这一波具有一种非常稳定和刻板的风格，这是所有SCN切片的共同特征。因此，它代表了一种保守的内部结构，但它的作用尚不清楚，尽管一些研究表明，它可能编码了昼长的季节性变化。我们希望了解它是如何产生的，然后我们将利用这一知识通过遗传和药理学方法来控制它，从而测试它的功能。为了实现我们理解SCN电路如何工作的总体目标，我们首先需要简化我们的分析，并专注于SCN细胞特定亚群的昼夜节律特性。然后，我们将看到相互连接(即组织成微电路)的细胞彼此之间的行为方式。为了实现这一点，我们将使用最先进的实时显微方法，并结合我们近年来开发的细胞群体的遗传靶向。这将使我们能够在单个SCN切片中跟踪几个昼夜节律周期，细胞电活动、钙水平(连接的一个特别重要的信号途径)和昼夜基因表达的节律。我们将重点放在细胞通过兴奋性神经肽或抑制性氨基酸GABA信号连接的微电路上。然后，我们将看到操纵细胞周期和/或肽能和GABA信号，以查看特定的微电路和整个网络发生了什么。这一建议将极大地促进我们对哺乳动物昼夜节律的神经基础的理解，也应该成为分析神经回路如何控制行为的范例。

Common experience tells us that we are 24h beings. Most obviously we sleep and wake on a daily cycle, and over the course of day and night our emotional state, cognitive abilities and general energy levels wax and wane on a regular programme. These rhythms are cued to the timing of sunrise and sunset, but when we travel between time-zones or work on rotating shift schedules we become aware that they are also dependent on internal cues. Indeed evolution has furnished us and almost all types of living organism with internal biological clocks that control our daily cycles, and are cued by sunlight. In mammals the major internal clock is the suprachiasmatic nucleus (SCN) of the brain's hypothalamus. It received nerve input from the eye to cues it to the cycle of dark and light but it does not need this input to generate its own cycles of neuronal activity which run with a period of approximately (circa-) one day (-dian): hence it is a "circadian" clock. One of the major advances in circadian biology over the past 20 years has been the identification of the genes and proteins that make up the circadian timer. In each cell of the SCN, these genes and the proteins that they encode are locked into a self-sustaining feedback loop of protein synthesis and followed by protein degradation. The biochemistry of these process means that the loop is completed approximately once a day. It is a cellular circadian mechanism that drives parallel circadian cycles of neuronal activity in the SCN, and these cycles of neuronal activity in turn communicate daily time to the rest of the brain and body. Despite this remarkable breakthrough in unravelling the "nuts and bolts" of the cellular clockwork, it remains many fundamental questions about the SCN unanswered. For example, the SCN consists of 20,000 cells and the clockwork in each is tightly coupled to that of the others. This is a necessary property if the SCN is to give a single unambiguous time signal to the body - but how is it achieved? Second, although they are synchronised, the clocks of the individual cells are not simultaneously active. When neuronal activity in the SCN cultured as an "organotypic" slice in a Petri dish is imaged microscopically, it can be seen that waves of activity sweep across the tissue on a daily basis. This wave has a very stable and stereotypical style, common to all SCN slices. It therefore represents a conserved internal structure to the circuit, but what it is for is not clear, although some studies suggest that it may encode seasonal changes in daylength. We wish to understand how it is generated, and then we shall use that knowledge to control it using genetic and pharmacological approaches and thereby test its function. To achieve our overall aim of understanding how the SCN circuit works, we first of all need to simplify our analysis and focus on the circadian properties of particular sub-populations of SCN cells. We shall then see how cells that are connected (i.e. organised into microcircuits) behave in relation to each other. To achieve this we shall use state-of-the-art real-time microscopic methods combined with genetic targeting of cell populations that we have developed over recent years. This will allow us to follow over several circadian cycles in single SCN slices cellular rhythms of electrical activity, calcium levels (a particularly important signalling pathway for coupling) and circadian gene expression. We shall focus on microcircuits where cells are connected by signalling by excitatory neuropeptides or the inhibitory amino acid GABA. We see shall then manipulate the cellular cycles and/ or peptidergic and GABA signals to see what happens to the particular microcircuit and to the overall network. This proposal will greatly advance our understanding of the neural basis of circadian timing in mammals and should also serve as an exemplar for the analysis of how neural circuits control behaviour.