Neuronal basis of ventilation in vertebrates

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Breathing is perhaps the most vital, robust and rhythmic of any vertebrate behavior. A neuronal circuit in the brainstem generates the respiratory rhythm and shapes the motor pattern of each breath to meet metabolic, biomechanical and behavioral goals. However, how this is achieved is not resolved. We have addressed these issues by focusing on the isolated bullfrog brainstem. This preparation produces breathing motor patterns resembling those of intact animals for at least 24 hrs. This resilience and the developmental accessibility of tadpoles makes frogs ideal for teaching neuroscience and for studying neuronal development and function in vertebrates. We discovered striking similarities in the neuronal circuits controlling breathing between bullfrogs and mammals despite fundamental differences in their breathing mechanics. We found two coupled, endogenously rhythmogenic neural networks (i.e., oscillators) in the isolated bullfrog brainstem, one for buccal ventilation, the other for lung inflation. Two brainstem oscillators in mammals have also been proposed, one for inspiration, the other for expiration. The putative oscillators in mammal and bullfrog occupy similar rhombomeric locations and appear to share several functional properties. Our recent data suggest a third oscillator in the frog brainstem, the ‘priming oscillator’ that appears responsible for the large buccal deflation that precedes lung inflation. This oscillator is distinct from the buccal oscillator and spans the lung oscillator occupying a much larger brainstem region.  These findings raise important fundamental questions: How well does the oscillator paradigm account for the complexity of the muscle recruitment pattern that occurs during each breath (timing of pump muscles and airway valves) in vivo? How are the oscillators connected? Do other air-breathing vertebrates have distinct oscillators for each phase of ventilation, and if so, how did they evolve? The objective of the proposed research is to expand understanding of breathing circuits by critically evaluating the oscillator construct as an underlying principle of respiratory rhythm generation in vertebrates. In the next 5 years, my trainees and I will: (1) Use standard neurobiological techniques to test the multiple oscillator paradigm in the decerebrate bullfrog and determine if oscillators alone account for the precise timing of pump-muscles and valve activation that occurs with each breath. (2) Record and label individual neurons within oscillators in isolated bullfrog brainstems to determine functional phenotypes, and use in situ hybridization to test for local expression of molecular markers that define mammalian respiratory rhythm generating neurons. (3) Use in situ hybridization for markers that demarcate oscillators in frogs and mammals to compare and contrast the architecture of breathing circuits in other lower vertebrates.