Modulation of arousal by direct CO2 gating of connexin hemichannels expressed by VTA GABAergic neurons

通过呼吸调节代谢废物二氧化碳（CO2）的清除对生命至关重要。如果过多的二氧化碳在血液中积聚，它就会变成酸性，这可能导致死亡。在过度换气和耐力运动期间，血液中的CO2水平会降低，并且会随着呼吸系统疾病（如哮喘）和吸烟而增加。我们最近产生的遗传证据表明，通过打开一个膜通道（由称为连接蛋白的蛋白质组成），可以直接在大脑中感知到升高的CO2，并且这在呼吸调节中起着重要作用。连接蛋白（Cx）联合收割机在细胞膜上形成大孔通道，介导细胞间通讯的重要方面。它们能够通过离子和小分子，如三磷酸腺苷（ATP）。连接蛋白可以以两种模式运作：i）两个相邻细胞中的通道可以对接在一起以形成细胞之间的通道-称为间隙连接;或者ii）它们可以简单地打开到细胞外的空间-称为半通道。有许多不同的连接蛋白（Cx）蛋白，但包括Cx 26的子集形成半通道，可以通过增加CO2打开。CO2直接与Cx 26结合，导致半通道打开。在脑干中，特定的神经胶质细胞表达Cx 26半通道，并且它们被高CO2打开导致呼吸的适应性变化，以将CO2水平降低到正常水平。我们最近发现，Cx 26半通道也表达的一个子集的神经元在大脑的一部分称为腹侧被盖区（VTA）。腹侧被盖区与情绪、动机和奖赏有关，也是控制睡眠和觉醒的神经回路的重要组成部分。一些腹侧被盖区神经元的激活促进睡眠，而它们的抑制促进唤醒。Cx 26半通道的表达使腹侧被盖区神经元直接对CO2敏感，更多的CO2降低了它们的兴奋性（当半通道打开时）。由于表达Cx 26的神经元促进镇静和睡眠，CO2的增加将关闭它们，这可能导致唤醒和觉醒。我们提出，腹侧被盖区这些特定神经元表达的Cx 26半通道检测到CO2水平的升高，并介导维持生命的反射：高碳酸盐唤醒。例如，在睡眠呼吸暂停中，当呼吸完全停止时，血液中二氧化碳水平升高会导致突然从睡眠中醒来，然后有意识地控制呼吸。有已知的机制如何提高二氧化碳可以导致觉醒，但这些依赖于血液pH值的变化（酸化），这只发生在大量的二氧化碳积累，因此发生缓慢。由于Cx 26半通道仅通过CO2的小幅增加而直接打开，因此我们假设它们将对CO2的相对小幅增加提供快速唤醒反应。为了探索我们的假设，我们将使用我们开发的遗传工具来选择性地从腹侧被盖区的神经元中去除Cx 26的CO2敏感性，然后测试对CO2水平变化的唤醒反应是如何改变的。我们预测这将减缓和钝化唤醒反应。然后，我们将确定CO2敏感的腹侧被盖区神经元如何连接到腹侧被盖区内的其他神经元群体和大脑中参与唤醒和清醒控制的其他核团。我们的项目将研究大脑中一个重要的神经元群体对二氧化碳的意外敏感性，这些神经元是多种复杂行为的控制中心。在这样做的过程中，我们将阐明唤醒如何被体内二氧化碳水平无意识地改变。

The regulated removal of the metabolism waste product carbon dioxide (CO2) via breathing is vital for life. If too much CO2 builds up in the blood it becomes acidic and this can lead to death. CO2 levels in blood will be reduced during hyperventilation and endurance sports and can increase with respiratory diseases such as asthma and with smoking. We have recently generated genetic evidence that raised CO2 can be directly sensed in the brain by opening a membrane channel (made up of proteins called connexins), and that this plays an important role in the regulation of breathing. Connexins (Cx) combine to form large-pored channels in the cell membrane to mediate important aspects of cell to cell communication. They are capable of passing ions and small molecules such as adenosine triphosphate (ATP). Connexins can operate in two modes: i) channels in two adjacent cells can dock together to form a passageway between the cells - called a gap junction; or ii) they can simply open into the space outside the cell - called a hemichannel. There are many different connexin (Cx) proteins, but a subset including Cx26 form hemichannels than can be opened by increases in CO2. CO2 binds directly to Cx26 to cause the hemichannels to open. In the brainstem, specific glial cells express Cx26 hemichannels and their opening by high CO2 causes an adaptive change in breathing to reduce the level of CO2 to normal. We have recently discovered that Cx26 hemichannels are also expressed by a subset of neurons in a part of the brain called the ventral tegmental area (VTA). The VTA is well documented to be involved in emotion, motivation and reward and is also an important part of the neural circuitry controlling sleep and wakefulness. Activation of some VTA neurons promotes sleep, with their inhibition promoting arousal. The expression of Cx26 hemichannels makes VTA neurons directly sensitive to CO2 with more CO2 reducing their excitability (as the hemichannels open). Since the neurons that express Cx26 promote sedation and sleep, increases in CO2 will switch them off and this could lead to arousal and wakefulness.We propose that Cx26 hemichannels expressed by these particular neurons in the VTA detect raised levels of CO2 and mediate a life-preserving reflex: hypercapnic arousal. For example, in sleep apnoea when breathing can stop altogether, raised levels of CO2 in the blood lead to abrupt waking from sleep followed by conscious control of breathing. There are known mechanisms for how raised CO2 can lead to arousal but these rely on a change in blood pH (acidification), which only occurs with the accumulation of significant amounts of CO2, and is thus slow to occur. As Cx26 hemichannels are directly opened by only small increases in CO2, we hypothesize that they will provide a rapid arousal response to relatively small increases in CO2. To explore our hypothesis, we will use the genetic tools that we have developed to selectively remove the CO2 sensitivity of Cx26 from the neurons in the VTA and then test how the arousal response to changing levels of CO2 is altered. We predict that this will slow and blunt the arousal response. We shall then determine how the CO2-sensitive VTA neurons connect to other neuronal populations within the VTA and other nuclei in the brain involved in arousal and control of wakefulness. Our project will study the unexpected sensitivity to CO2 of an important population of neurons in the brain that are a control hub for multiple complex behaviours. In doing so, we will shed light on how arousal may be unconsciously modified by the level of CO2 in the body.