We have developed an in vitro model for studying electrophysiological properties and intracellular signalling in identified neuronal subpopulations, in particular GABAergic and noradrenergic (NAergic) neurones in the rat brainstem. To specifically visualise the neuronal phenotypes of interest, we use adenoviral vectors which express fluorescent proteins (such as EGFP) under control of cell-specific promoters. For targeting GABAergic neurones we use a 3.7kB part of the GAD67 promoter; NAergic neurones are targeted by the short synthetic PRSx8 promoter (see Teschemacher et al. 2005). Sterile brainstem slices from humanely killed rat pups (p9-11) are transduced with viral vectors and cultured on Millipore organotypic membranes as previously described (Teschemacher et al. 2005). After 7-14 days, slice cultures are transferred into a glass bottomed recording chamber which is mounted on the stage of an upright laser-scanning confocal microscope equipped with DIC optics (Leica SP). The cultures are continuously superfused at 31±1°C with artificial cerebrospinal fluid. EGFP-positive neurones are approached with patch pipettes filled with a solution containing the red-shifted Ca²⁺ indicator Rhod-2 (0.5 mM). Diffusion of EGFP into the recording pipette is visualised using the 488-nm wavelength line of an argon laser and confirms the successful establishment of whole-cell configuration with the targeted neurone. About 20 minutes after seal rupture, neurones are sufficiently loaded with Rhod-2 for measurements of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) to commence. Rhod-2 is excited at 543 nm with a He-Ne laser and emitted light is sampled within the 560–640-nm band. Two-dimensional images can be scanned at 1-10 Hz. For higher temporal resolution, line scans focused on an area of particular interest can be employed at a frequency of about 2 kHz. For experiments with the green-emitting Ca²⁺ dyes, such as Oregon Green or Fluo 4, a viral vector expressing monomeric red fluorescent protein can be used. We observe changes in [Ca²⁺]_i following electrical stimulation of the neurone via the recording pipette or by adding modulators of intracellular signalling cascades (such as nitric oxide donors or angiotensin II) to the bath solution. We analyse images off-line to extract information on differential [Ca²⁺]_i changes in subcellular compartments such as soma, dendrites, and putative axons and transmitter release sites. This technique enables us to directly study the details of intracellular signalling and electrical activity of defined neuronal phenotypes in vitro at high spatial and temporal resolution.