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

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.

Hemichannels composed of connexin 26 (Cx26) are directly gated by CO2, being opened or closed by modest changes in PCO2 around normal levels. We have developed a dominant negative (DN) viral construct that allows us to reduce the CO2 sensitivity of Cx26 hemichannels without changing hemichannel expression. We have delivered this DN construct to a confined target area of the medulla oblongata and shown that CO2 binding to Cx26 hemichannels in a population of glial cells contributes about half of the adaptive ventilatory response to hypercapnia controlled by central chemosensors.

We have recently discovered that CO2-sensitive Cx26 hemichannels are not only expressed by glial cells but surprisingly are also expressed by a specific set of central neurons in the ventral tegmental area (VTA) in the midbrain. These neurons are GABAergic and the opening and closing of the Cx26 hemichannels by modest changes in CO2 markedly changes their conductance and excitability. The very precise localisation of Cx26 in this group of neurons suggests they have physiological importance.

The VTA is a critical component of the sleep/wake circuitry and we therefore propose that the expression of CO2-sensitive Cx26 hemichannels in VTA GABA neurons plays a major role in hypercapnic arousal. To test this, we will use the genetic tools that we have developed (dnCx26) together with cre mouse lines to selectively reduce CO2 sensitivity of Cx26 hemichannels in specific neuronal populations. We will assess the effect this has on the arousal response to hypercapnia (using EEG and EMG readouts). Using retroviral tracing together with cre mouse lines we will begin to define this novel VTA hypercapnic arousal circuit.

We hypothesise that Cx26 hemichannel expression in the VTA provides a rapid arousal pathway, when CO2 is raised for example in conditions such as sleep apnoea. Our work will define a new role for the VTA, a major hub for controlling complex behaviours.