Central respiratory chemosensitivity is crucial to maintain constant levels of PCO2 and pH within the brain. The retrotrapezoid nucleus (RTN), located close to the ventral surface of the medulla oblongata, contains a population of neurones previously proposed to act as primary central chemoreceptors (1). Astrocytes provide structural and metabolic support for neurones, but direct evidence indicating their role in complex behaviours is limited. Our hypothesis is that pH-sensitive astrocytes located in the ventral medulla oblongata are the actual central chemoreceptors that activate RTN neurones via release of ATP. To test it we have generated a novel viral vector Ad.SuperI.PRSx8.TN-XXL which expresses a calcium sensor, TN-XXL (2), under the control of the enhanced PRSx8 promoter. PRSx8 is activated by the transcription factor Phox2b which is, in this region, selectively expressed in RTN neurones (1). Promoter activity was enhanced by two-step transcriptional amplification (3), whereby the co-expressed recombinant transactivator, GAL4BDp65, binds to a GAL4 binding sequence placed upstream of PRSx8 and thus potentiates the expression of TN-XXL. The function of TN-XXL is based on Föster resonance energy transfer (FRET) between two genetically encoded fluorescent proteins, CFP and YFP. Binding of Ca2+ leads to a shift in YFP/CFP fluorescence ratio (2). Organotypic slices (from Wistar pups p8-10) including the RTN region were prepared using methods described earlier (4) and transduced with Ad.SuperI.PRSx8.TN-XXL. After 7 days slices were superfused with HEPES-buffered saline (33οC) in a tissue chamber and imaged using a Zeiss 510 CLSM upright confocal microscope. Fluorescence was excited with a 456 nm argon laser and collected using 480-520 nm and 535-590 nm filters for CFP and YFP, respectively. KCl (25mM) was used as a positive control to obtain a maximal increase in the YFP/CFP ratio during neuronal depolarisation (198.8±9.5%; n=8; Student’s paired T-Test p<0.01). A shift of pH from 7.4 (baseline) to 7 induced a 21.3±4.1% Ca2+ increase (n=16; p<0.01). However, in the presence of the P2Y receptor blocker MRS2179 (3mM; IC50= 0.3μM), acidification did not induce Ca2+ elevations (5.1±1.8%; n=8; p=0.01). These observations suggest that pH-induced activation of RTN neurones depends on prior release of ATP which was previously demonstrated to occur in response to acidification and hypercapnia in this area (5). Thus, we have generated a novel imaging tool which will be useful for inquiries into the properties of RTN neurones and other neuronal populations where the PRSx8 promoter is active.