Functional properties, neurochemistry and connectivity of medullary neural networks that control breathing and circulation

The autonomic nervous system governs basic homeostatic functions such as regulating cardiac output, blood pressure, body temperature, and the generation of respiratory rhythm. Although traditionally conceptualised as independent entities, the neural networks that underlie respiratory rhythm and vasomotor tone are intimately connected, with each component influencing the activity of the other. This respiratory-sympathetic coupling contributes to cardiac sinus arrhythmia and underlies the strong respiratory modulation of vasomotor sympathetic nerve activity, which may play an important role in initiation and development of neurogenic hypertension. The experiments described in this thesis explore the neurochemical profiles, functional properties, and circuit architecture of medullary neurons that control breathing and circulation. Genetically modifying a single functionally identified neuron in an intact neural network would provide the ultimate way to bridge neuronal circuit structure and function. The work described in Chapter 2 of this thesis describes a novel method that combines extracellular recording with the labelling of neurons recorded in the ventral lateral medulla with either dyes or plasmid DNA, permitting conventional cell labelling or gene delivery. We demonstrate that our approach has several advantages over traditional single-cell labelling approaches, and therefore provides a useful tool for correlation of functional properties of single neurons with their neurochemical phenotypes that can also be used for targeted single cell gene delivery under blind recording conditions. We then apply this technique to address a controversial topic in the field of respiratory neuroscience; the neurochemical signature of the neurons responsible for generating respiratory rhythm. Previous investigations have suggested that the neuropeptide somatostatin plays a critical role in respiratory rhythmogenesis and is a marker for excitatory respiratory interneurons in the pre-Bötzinger complex (preBötC). Somatostatin receptor type 2a (sst₂ₐ) has been postulated as a mediator of such effects, and has also been proposed as a marker for respiratory neurons. In Chapter 3 we systematically examine the expression pattern of sst₂ₐ with regards to the functionally distinguished subcomponents of the ventral respiratory column. We further examine the feasibility of sst₂ₐ as a reliable marker of respiratory function at the single cell level. In Chapter 4 we examine the anatomical substrate responsible for linking the respiratory and sympathetic circuits using a modified trans-synaptic tracing tool. We first extensively map sources of monosynaptic drive to putative sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) using a genetically restricted rabies variant, that is selectively directed towards RVLM neurons that express the Phox2b transcription factor. We identify likely sources of inputs within brainstem compartments with proven roles in generation of respiratory rhythm and phase transition in normotensive animals. The data from this thesis extends our knowledge of respiratory neuroanatomy and provides important clues to the organisation of the circuits that underlie respiratory-sympathetic coupling.