Unbuckling the Buccal oscillator in the American Bullfrog

Abstract

The American Bullfrog is a remarkable model organism to study rhythm generation underlying breathing. It exhibits a breathing pattern that can be divided into four distinct ventilatory phases: lung‐priming, lung‐powerstroke, buccal‐dilation, and buccal‐constriction. We recently established that lung‐priming and lung‐powerstroke are generated by two separate burst generating substrates in the brainstem—the Priming area and Powerstroke area—located in rhombomere 4–5. The neural origin of the two phases of buccal, on the other hand, remains elusive. Our previous experiments suggest that a Buccal area located in rhombomere 7 is responsible, at least in part, for generating the biphasic buccal rhythm; however, whether any additional burst generating substrates are involved is yet unknown.The objective of this study was to characterize areas in the frog brainstem that generate the biphasic buccal rhythm. We utilized the isolated frog brainstem preparation and recorded fictive respiratory motor output from the trigeminal and facial cranial nerves using extracellular suction electrodes. We progressively transected the brainstem in both directions (caudal‐to‐rostral, and rostral‐to‐caudal) to identify regions where transection eliminated one or both buccal burst phases.Our preliminary results suggest that both phases of the buccal rhythm are generated by substrates located at the same level as the previously identified Buccal area. Nevertheless, using our current methodology, we are unable to discern whether multiple substrates or a single substrate is generating the buccal rhythm. To address this question, we developed a ‘buccal slice’ preparation at the level of the Buccal area which, when exposed to 100nM AMPA, produces buccal‐like bursts. We continue to develop this preparation as it holds promise for further dissecting the properties of this system and exploring it at the cellular and molecular levels.Our analysis suggests that the Buccal area is an evolutionary homolog of the mammalian preBötzinger complex. Both are located in rhombomere 7, are resilient oscillators, being staunchly insensitive to CO2, and co‐opted for sniffing. We anticipate that having two comparable preparations, i.e. ‘buccal slice’ and ‘preBötC slice’, will allow for more direct comparisons between these oscillators, further testing their homology and thus take us closer towards understanding the evolution of breathing.Support or Funding InformationNational Science and Engineering Council of Canada (NSERC) and Alberta Innovates Health Solutions (AIHS) provided funding for this study.