The characteristics of noradrenaline (NA) release from central neurones have largely been inferred from experiments in model systems such as bovine adrenal chromaffin cells or the PC12 cell line (Ninomiya et al. 1997;Teschemacher & Seward, 2000). In these models it is possible to place microamperometric electrodes on individual cells and monitor exocytosis of NA. Similar experiments in native brain noradrenergic (NAergic) neurones have not been possible because the release sites are difficult to visualise and access. We have developed a method which allows direct recording of NA release from NAergic neurones from specific brainstem nuclei.

To this end organotypic brainstem slice cultures are prepared from humanely killed 9-day-old rats. Cultures are subsequently transfected with an adenoviral vector which causes expression of EGFP selectively in NAergic neurones under the control of an artificial promoter (PRSx8; Hwang et al. 2001). After >3 days, clusters of fluorescent neurones can be visualised using conventional epifluorescence and confocal microscopy in living slices in the areas consistent with the location of the known brainstem NAergic and adrenergic cell groups (A1, A2, A6, C1). In addition to cell bodies, multiple processes including characteristic beaded axons can be traced for hundreds of micrometers. These axons were approached by microamperometric probes (5 µm tip diameter). Oxidative current spikes were detected as described for vesicular catecholamine release in isolated cell models. This method will be used to study modulation of NA release from the different cellular compartments of identified NAergic neurones and can be combined with further genetic manipulation of the same cells.

This work was supported by the Royal Society (23697), and BBSRC (7/JE616459)