In 2005 Gourine et al (Gourine et al., 2005) demonstrated that ATP is released at the ventral surface of the medulla oblongata during CO2-evoked activation of the central chemoreceptor area. It was also found that ATP release constituted an important initial event in central chemosensory transduction process. We hypothesised that ATP could be released from the local chemosensitive astrocytes and that these cells constitute an important link in this elusive mechanism. To study astrocyte activity in vitro and in vivo we expressed a genetically encoded Ca2+ indicator Case12 in ventral medullary astrocytes from Sprague Dawley or Wistar rats using viral vectors. Mild acidification in vivo (under α-chloralose anaesthesia (100 mg kg-1 iv, supplemented with 20 mg kg-1 iv as required following femoral vein cannulation under 3% isoflurane induction) and in vitro triggered ATP-mediated Ca2+ waves in astrocytes located at the ventral edge of the medulla. Acidification-induced Ca2+ responses were abolished in the presence of bafilomycin A (blocker of vesicular pumps) and brefeldine A (blocker of vesicular transport) indicating that ATP is released from a vesicular pool. In vitro, ATP receptor antagonists reduced electrophysiological and Ca2+ responses of the local population of chemosensitive neurones (of the retrotrapezoid nucleus, RTN) to changes in pH, suggesting that ATP-mediated signalling plays a key role in central chemosensory transduction. In order to selectively stimulate astrocytes we expressed in these cells a light-sensitive non-selective cation channel ChR2. Stimulation of transduced astrocytes with blue light triggered Ca2+ elevations and release of ATP as measured by luciferase assay. Light activation of astrocytes in the central chemosensitive area (overlapping with RTN) led to activation of the respiratory activity in vivo and this effect could be blocked by MRS2179 an antagonist with preferential tropism to P2Y1 and P2X1 receptors. These experiments demonstrated the involvement of astroglial purinergic signalling mechanisms in one of the most fundamental physiological processes in the CNS(Gourine et al., 2010). To further study astrocyte-to neuron signalling, we have generated vectors to express light-sensitive G-protein coupled receptors (OptoAlpha1- and OptoBeta2 )(Airan et al., 2009) selectively in astrocytes using vectors with enhanced GFAP promoter(Liu et al., 2008). Both OptoAlpha1 and OptoBeta2 vectors were studied in vitro and we have verified that OptoAlpha1 signals via PLC and IP3 while OptoBeta2 signals via adenylate cyclase. Light activation of OptoAlpha1—expressing astrocytes in the rostral-ventro-lateral medulla (RVLM) evoked powerful pressor responses and increases in renal sympathetic nerve activity. Similar responses are evoked following activation of ChR2 expressed in RVLM glia. The signalling mechanisms used by astrocytes in RVLM are currently under investigation. In summary, optogenetic approaches allow interrogation of the intimate mechanisms underlying astrocyte-neuronal signalling in vitro and in vivo and demonstrate that purinergic signalling plays an important role in astrocyte-neuronal communication in brainstem autonomic structures.