Key pointsr For most multiphasic motor patterns, rhythm and pattern are produced by the same circuit elements. For respiration, however, these functions have long been assumed to occur separately.r In frogs, the ventilatory motor pattern produced by the isolated brainstem consists of buccal and biphasic lung bursts. Previously, two discrete necessary and sufficient sites for lung and buccal bursts were identified.r Here we identify a third site, the Priming Area, important for and having neuronal activity correlated with the first phase of biphasic lung bursts.r As each site is important for burst generation of a separate phase, we suggest each major phase of ventilation is produced by an anatomically distinct part of an extensive brainstem network.r Embedding of discrete circuit elements producing major phases of respiration within an extensive rhythmogenic brainstem network may be a shared architectural characteristic of vertebrates.Abstract Ventilation in mammals consists of at least three distinct phases: inspiration, post-inspiration and late-expiration. While distinct brainstem rhythm generating and pattern forming networks have long been assumed, recent data suggest the mammalian brainstem contains two coupled neuronal oscillators: one for inspiration and the other for active expiration. However, whether additional burst generating ability is required for generating other phases of ventilation in mammals is controversial. To investigate brainstem circuit architectures capable of producing multiphasic ventilatory rhythms, we utilized the isolated frog brainstem. This preparation produces two types of ventilatory motor patterns, buccal and lung bursts. Lung bursts can be divided into two phases, priming and powerstroke. Previously we identified two putative oscillators, the Buccal and Lung Areas. The Lung Area produces the lung powerstroke and the Buccal Area produces buccal bursts and -we assumed -the priming phase of lung bursts. However, here we identify an additional brainstem region that generates the priming phase. This Priming Area extends rostral and caudal of the Lung Area and is distinct from the Buccal Area. Using AMPA microinjections and reversible synaptic blockade, we demonstrate selective excitation and ablation (respectively) of priming phase activity. We also demonstrate that the Priming Area contains neurons active selectively during the priming phase. Thus, we propose that three distinct neuronal components generate the multiphase respiratory motor pattern produced by the frog brainstem: the buccal, priming and powerstroke burst generators. This raises the possibility that a similar multi-burst generator architecture mediates the three distinct phases of ventilation in mammals.M.I. Baghdadwala and M. Duchcherer contributed equally to this work.

Central pattern generators located in the brainstem regulate ventilatory behaviors in vertebrates. The development of the isolated brainstem preparation has allowed these neural networks to be characterized in a number of aquatic species. The aim of this study was to explore the architecture of the respiratory rhythm-generating site in the goldfish (Carassius auratus) and to determine the utility of a newly developed isolated brainstem preparation, the Sheep Dip. Here we provide evidence for a distributed organization of respiratory rhythm generating neurons along the rostrocaudal axis of the goldfish brainstem and outline the advantages of the Sheep Dip as a tool used to survey neural networks.

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