Characterisation of Flower-Spray Endings of the Cardiac Atria of the Rat using a robotic system

Background: Atrial volume receptors are neurons whose mechanically sensitive endings are located in the cardiac atria, predominantly at the cavo-atrial junctions. These mechanotransductive neurons form the afferent limb of the atrial volume reflex which contributes to plasma volume regulation. Atrial volume receptors are classified as type A, B, or intermediate depending upon the timing of their activation with respect to the atrial cycle. Type A receptors activate during atrial systole, type B receptors during atrial diastole and intermediate receptors activate during both atrial systole and diastole. Histological examinations of the subendocardial neural plexus have identified two endings as candidate mechanoreceptors: flower-spray endings (also known as complex unencapsulated endings) and end-net formations. The aims of this thesis were to map the receptive field of atrial volume receptors using ex-vivo models of the Wistar rat right atrium, to characterise the neural plexus and nerve terminals present within, as such investigations could either support or refute the hypothesis that flower-spray endings and / or end-net formations are the mechanotransductive terminals of the cardiac atria. In addition, it was aimed to compare the properties of atrial mechanoreceptors with baroreceptors of the aortic arch and the muscle spindle of skeletal muscle. Methods: From ex-vivo models of Wistar rat tissue, afferent neuronal activity from mechanically sensitive neurons was recorded using a glass suction microelectrode technique and spike filtering software. An existing XYZ robotic system was upgraded to a multitool machine, permitting both the application of finely controlled stimuli to mechanotransductive tissues and epifluorescence imaging of atrial tissue. The XYZ robotic system was integrated with a neurophysiology data recording system to facilitate the exchange of data between microcontrollers. Automated receptive field mapping protocols were created to visualise coordinate and nerve activity data in the form of heat maps and these mapping protocols were applied to a new ex-vivo model of the right atrium termed the “mapping” model. Atrial mechanoreceptor receptive field data was presented as dynamic and static stimulus response heatmaps. A FM1-43 labelling protocol was developed to label the right atrial subendocardial neural plexus and characterise the neural formations contained within mapped receptive fields. Findings: The “mapping” model of the right atrium permits automated mapping of atrial mechanoreceptor receptive fields which are mostly located in the posterior wall between the mid-atrial region and inferior cavo-atrial junction. Atrial mechanoreceptors may possess a single receptive field or multiple small non-overlapping receptive fields. There is no statistically significant different in the mean receptive field size between units that have a single receptive field and those that have multiple small non-overlapping receptive fields. FM1-43 labelling of the receptive field rich zone consistently revealed a rich neural plexus in the subendocardial space which contained neural structures similar to end-net formations and unencapsulated endings. Conclusions: The receptive field of atrial mechanoreceptors can be mapped using an automated robotic system which when combined with FM1-43 neural labelling, permits characterisation of the sensory terminals contained therein. As it is unknown whether the observed FM1-43 labelled subendocardial neural formations were selectively labelled via mechanotransductive processes, additional investigations are required to determine their ability to encode physical stimuli. The receptive field mapping and FM1-43 labelling protocols established in this thesis comprise a significant undertaking towards these clinch experiments. Given their great clinical importance, further investigations into the molecular basis of atrial mechanotransduction are warranted.