CO2 sensitive connexin channel synapses in the VTA release 5HT to regulate dopaminergic neurons

It is now well established that the major mid-brain dopaminergic center, the ventral tegmental area (VTA), plays an important role in the control of sleep-wake state transitions, producing arousal from sleep states upon activation. We recently showed that the VTA may also be involved in producing arousal in response to hypercapnia, a key survival response. We found that connexin 26 (Cx26) CO2-sensitive hemichannels are expressed by VTA GABAergic neurons and modulate their excitability. Here we have extended our investigation of VTA chemosensing and have discovered additional novel mechanisms of CO2 neural signalling: connexin co-synapses. CO2-sensitive hemichannels are expressed on the cell bodies and the terminals of dorsal raphe (DR) serotonergic neurons that cluster around VTA TH+ dopaminergic neurons colocalizing both with the 5HT transporter SERT and with VGLUT3. Using pharmacological dissection and GRAB5-HT sensors, we showed that 5HT can permeate through open Cx26 hemichannels to modulate the excitability of VTA dopamine neurons. Brief elevations of pCO2 regulate dopaminergic neuron excitability via 5HT3 and 5HT2A/C receptors activation. Longer applications of raised pCO2 modulates HCN channel opening in response to hyperpolarizing current steps, an effect blocked by 5HT2 receptor antagonists. These effects of pCO2 can be blocked with the connexin inhibitor lanthanum. In vivo, CO2 challenge (hypercapnia) activates VTA neurons (including dopaminergic neurons), with the activation greatly reduced by the conditional knockdown of Cx26 expression in the VTA. We therefore propose that the VTA could play an important role in hypercapnic arousal with at least two distinct mechanisms of CO2 signalling: modulation of GABAergic neuron excitability via soma Cx26 hemichannels and channel-mediated release of 5HT via CO2 sensitive Cx26 co-synapses on DR nerve terminals that regulate the excitability of dopaminergic neurons.