One role of noradrenergic (NAergic) neurotransmission in the medulla oblongata is in the regulation of arterial pressure (Reis et al. 1977; Duale et al. 2005). In order to gain a better understanding of the mechanisms regulating central noradrenaline (NA) release, we imaged intracellular Ca²⁺ signalling in NAergic axonal varicosities of A1 and A2 neurones. Organotypic rat brainstem slice cultures were transfected with adenoviral vectors driving expression of enhanced green fluorescent protein in catecholaminergic neurones specifically (Teschemacher et al. 2005a). Green fluorescent NAergic neurones were visualised (excitation 488, emission 500-530 nm) by combined upright confocal and DIC optics on a Leica SP2 system. Axonal varicosities (diameters ~2-4 μm) were evident on axonal branches several hundred micrometers from their cell body. This distance precluded adequate filling with a fluorescent Ca²⁺ indicator loaded from a patch electrode located at the soma (Teschemacher et al. 2005b). Therefore, we patched varicosities directly and loaded them with the Ca²⁺-sensitive dye Rhod-2 (0.5 mM) contained in the pipette solution. Changes in their intracellular Ca²⁺ concentration were monitored by confocal fluorescence imaging (excitation 543 nm, emission 560-630 nm) in XY-t mode (at ~ 15 Hz) or in X-t line scan mode (at ≤ 1 ms/line). We found that adjacent varicosities were loaded successfully after patching a single varicosity. Electrical recording duration was variable (10-60 min). With good electrical access, action potentials were triggered by injection of current through the patch pipette. In the absence of electrical access, the axon was stimulated electrically via an extracellular microelectrode. Both modes of electrical stimulation evoked Ca²⁺ transients. In addition, Ca²⁺ transients also occurred spontaneously without evidence of action potentials. In conclusion, we have developed an approach for imaging Ca²⁺ transients in NAergic axonal varicosities. The finding of action potential-independent Ca²⁺ transients suggests the local activation of metabotropic receptors which may mediate Ca²⁺ release from intracellular stores in varicosities.