An Isolated Atrial Preparation to Study the Molecular Mechanisms of Vagal Volume Receptors

Background: Atrial volume receptors are a significant reflexogenic zone of the circulation and are located in the subendocardium of the cavo-atrial and atrio-pulmonary venous junctions. In previous histological studies, the atrial volume receptors were classified according to the form of the sensory endings into two types: end nets and flower sprays. However, the significance of the two morphologies as well as the molecular mechanisms of mechanotransduction that atrial volume receptor utilise remain to be elucidated. The aim of this thesis was to establish a working isolated atrial preparation with intact vagal function that permits molecular analysis of mechanotransduction. A search was conducted for a fluorescent vagal sensory label that can be applied to a living preparation with retention of vagal function. Methods: The viability of vagal function in ex-vivo open atrial preparations was examined using stimulation and recording protocols involving the cardiac branch of the vagus nerve. Orthodromic electrical stimulation of preganglionic vagal fibres was combined with afferent recording through a common head stage. A number of fluorescent dyes were examined to visualise the subendocardial neural plexus, including the styryl dye FM1-43 and the calcium indicators Fluo-4 AM and Oregon Green 488 BAPTA-1 AM. Findings: Superior and more extensive labelling of the subendocardial neural plexus was exhibited by FM1-43 compared to Fluo-4 AM and Oregon Green 488 BAPTA-1 AM. It successfully labelled nerve fibres with the classic appearance of end nets and flower sprays at the inferior cavo-atrial junction. Oregon Green 488 BAPTA-1 AM was shown to selectively stain the nuclei of endocardial cells. Ex-vivo open atrial reparations at room temperature show a more obvious vagally mediated chronotropic and inotropic response than warmed preparations. This relates in part to the negative Treppe effect that the rat heart exhibits. Using a fixed stimulation duration, the optimal frequency for negative inotropy was 16 Hz while that for negative chronotropy was 8 Hz. Both mechanosensitive and chemosensitive vagal sensory single units were also recorded
in the isolated atrial preparation at room temperature. Conclusions: These findings have shown FM1-43 to be the label of choice for use in future studies of the subendocardial plexus and the molecular mechanisms of atrial volumereceptors. The isolated atrial preparation at room temperature retains both motor and sensory vagal function and is considered a suitable model for neuroanatomical investigations of rat atrial volume receptors.