Glia-neuronal interactions are essential for normal functioning of the nervous system during development and adult life. However many aspects of these interactions are still unclear. By releasing glio-transmitters, glia can affect neuronal excitability and synaptic transmission and potentially coordinate activity across networks of neurones. Selective activation of astrocytes is now possible by optogenetic approach. Here, we compare signalling mechanisms between astrocytes and two types of neurones, the noradrenergic (NAergic) neurones in the pontine locus coeruleus (LC) and glutamatergic neurones in the retrotrapezoid nucleus (RTN) located near the ventral surface of the medulla oblongata. Experiments were performed in organotypic slice cultures of Wistar rats. Astrocytes were transduced with a novel adenoviral vector AVV-sGFAP-ChR2 (H134R)-Katushka1.3. This vector contains an enhanced glial acidic fibrillary protein promoter which is highly selective for astroglia (Liu et al., 2008). It expresses a mutated version of the light-sensitive channelrhodopsin 2 (ChR2-H134R33) fused with a far-red shifted novel fluorescent protein Katushka1.3 (Gradinaru et al., 2007; Shcherbo et al., 2009). We first patched the AVV-sGFAP-ChR2(H134R)-Katushka1.3-transduced astrocytes and found that in these cells blue light induced precisely timed depolarisations (+ 18.8 ± 2.0 mV, n=7, p<0.0001). Both, RTN neurones and NAergic neurones in LC, were visualized using adenoviral viral vectors with PRSx8 promoter to express DsRed-2 fluorescent protein. Patch clamp recordings were made from DsRed2-positive neurones in LC and RTN. Activation of astrocytes with blue light evoked long lasting depolarisations of adjacent RTN neurones (+ 3.57 ± 0.5 mV, n=9, p<0.001) and LC neurones (+ 3.08 ± 0.33 mV, n=14, p<0.001). These responses in RTN neurones were blocked in the presence of the ATP receptor antagonist MRS 2179 (10 μM, depolarisation + 0.39 ± 0.06 mV, n=7, p<0.001), but both MRS 2179 (10 μM) and another ATP receptor antagonist TNP-ATP (10 μM) had no effect on depolarisation of LC neurones evoked by light activation of astrocytes (n=6, p=0.71 for MRS2179; n=9, p=0.86 for TNP-ATP). However, astrocyte-evoked depolarisation of LC neurones was suppressed by NMDA receptor antagonist APV (50 μM, depolarisation + 0.84 ± 0.66 mV, n=5, p<0.01) and AMPA receptor antagonist CNQX (10 μM, depolarisation + 0.64 ± 0.35 mV, n=5, p<0.001). We conclude that activated astrocytes signal to RTN neurones via an ATP-dependent mechanism. In contrast, in LC, astrocytes are able to activate NAergic neurones by releasing glutamate. These data imply that glia-neuronal signalling occurs in an area-specific and transmitter-dependent manner.