Voltage-sensitive dyes can be used to monitor changes in membrane excitability from neuronal networks (Briggman et al. 2005; Onimaru & Homma, 2003; Tominaga et al. 2000). The information gleaned gives both temporal and spatial patterns of activity. In the present study we applied voltage-sensitive dyes to the mammalian respiratory network to assess the feasibility of this technique. Our initial question was to assess whether there was any spatio-temporal differences of neuronal activity within the ventral respiratory group (VRG) between eupnoea and gasping. Rats (Wistar; male; 80-100g) were anaesthetised deeply in 4% halothane. Once there was an absence of a withdrawal response to noxious pinching of the tail or a paw, an arterially perfused in situ working heart-brainstem preparation was prepared. This has good mechanical stability of the brainstem and light scattering is minimised by using a cell-free perfusate. Using a lateral approach the ventrolateral medulla was exposed (see Dutschmann & Paton, 2003). Contained within a glass micropipette di-2-ANEPEQ (50μg/ml; Molecular Probes) was microinjected into the VRG as defined by mass respiratory related activity recorded extracellularly. Neuronal activity was recorded using an optical recording system (MiCAM02 system, Brain Vision Inc., Tsukuba, Japan) through a 510-550 nm excitation filter, a dichroic mirror and a 590 nm absorption filter. Illumination was provided by a tungsten-halogen lamp (150 W) and a CCD camera (180 × 120 pixels) with a maximum time resolution of 3.5 ms was used to detect a change in fluorescence of the voltage-sensitive dye. Changes in fluorescence (F) were measured as ΔF/F and expressed as au. Magnification was provided by a ×2 objective (Olympus Optical) that resulted an imaging area of 4.2 × 3.25 mm². Phrenic nerve related patterns of neuronal activity were seen throughout the VRG (n=4); these included regions exhibiting inspiratory (0.66±0.07) and post-inspiratory (0.27±0.02) related changes in fluorescence. During gasping (induced with 7% oxygen; n=4), activity was confined to a restricted region that exhibited inspiratory related changes in fluorescence only (2.54±0.5). We conclude that voltage-sensitive dyes can be used in situ and that temporal and spatial information can be obtained from the VRG under conditions of both eupnoea and gasping.