Project Outline

Breathing is our first act upon birth, and the last action we complete before death. The first to last breath taken, is in fact, how we define someone's life. We don't love our children from their 'first blink', we don't love our partners until our 'dying thought', soldiers do not fight until their last 'heart beat', and nobody at a surprise encounter has ever said 'as I live and walk'. However we risk taking breathing for granted. The breathing pattern is generated by specialised nuclei -the preBötzinger complex (preBötC; controlling inspiration), a post-inspiratory oscillator, currently thought to be the postinspiratory complex (PiCo), and the parafacial lateral region (pFL: controlling expiration).

We know a significant amount about the inspiratory oscillator: It's subdivisions, as well as it's inputs and outputs; We even know it's human analogue, which consists of ~120,000 neurons. Much less is known about the other 2 oscillators. Of the remaining 2, the expiratory oscillator is the better studied but even then our knowledge is minimal: We know broadly which neurotransmitters these neurons produce, but not if they are sub-divided further; we know the function of the excitatory subtype, but not the inhibitory one, and whilst we have a map of it's inputs, it's outputs remain largely unknown. Finally, the post-inspiratory oscillator remains largely uninvestigated; we know it's subdivisions but the function of them is mostly undetermined, other than they drive post-inspiratory motor output whilst reducing inspiratory period; we know little about it's outputs except that it does not appear to project directly to the expiratory oscillator.

In this set of experiments we will manipulate different subpopulations of neurons in the rhythm generating network. The manipulations will focus on the role of the often overlooked inhibitory neurons, and the excitatory neurons that drive them. By changing their activity individually or in combination, we can begin to parse out how the different types of neurons in these oscillators work together to pattern motor output. Furthermore, we will make the neurons express fluorescent molecules to identify and quantify the neurons, whilst simultaneously allowing us to build an anatomical map of the outputs of these neurons, upon which our functional data will be added. By building such an in depth map we hope to build a model of respiration to help guide future experiments and therapeutic targeting.

Anatomical mapping and study of functional interactions that generate the phased motor output of breathing are vital areas in need of research. We have already begun this work, by dissecting the functional interactions of the inspiratory (preBötzinger Complex: preBötC) and expiratory (parafacial lateral region: pFL) oscillators. Using a urethane anaesthetized spontaneously breathing rat preparation, we will extend this to the subpopulation level of the network, and expand the model to include a third oscillator that controls post-inspiration (postinspiratory complex: PiCo), and a known inhibitory nucleus whose primary role is to coordinate the phases of breathing, the Bötzinger Complex (BötC). Given the duration of the project we will focus on how inhibitory neurons within the preBötC, BötC, and pFL, and the excitatory neurons within preBötC, PiCo, and pFL that drive them, to see how they pattern breathing, and particularly expiration. Using commercially available viral plasmids, our collaborator will supply us with all of the necessary viruses to complete this project. We will utilize well established optogenetic (opsins) and chemogenetic tools (Designer Receptors Exclusively Activated by Designer Drugs (DREADDS) and Pharmacologically Selective Actuator Modules (PSAMs)), to simultaneously alter the output of 2 different sub-populations of the rhythm generator. This functional map of interactions, will be enhanced by the simultaneous building of an anatomical map of anterograde connections of these nuclei. At the end of this study we will have created the most in depth map of the neural microcircuit for expiratory related breathing and created a series of tools to functionally probe them.

Further Reading

Chemogenetic manipulation: https://elifesciences.org/articles/14203

Optogenetic manipulation: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340826/

Techniques

• Optogenetics
• Chemogenetics
• Plethysmography
• Non-recovery surgery
• Recovery surgery
• Immunocytochemistry
• Epifluorescent/confocal microscopy